Molecular characterization with carbon nanotube control

ABSTRACT

There is provided a first reservoir containing a liquid solution including a molecule to be characterized and a second reservoir for containing a liquid solution including a molecule that has been characterized. A solid state support structure is provided including an aperture having a molecular entrance providing a fluidic connection to the first reservoir and a molecular exit providing a fluidic connection to the second reservoir. One carbon nanotube is provided having a longitudinal sidewall disposed as a molecular contacting surface at the aperture. A voltage source is connected in series with the carbon nanotube for electrically biasing the carbon nanotube, and an electrical current monitor is connected in series with the carbon nanotube for monitoring changes in electrical current through the nanotube corresponding to translocation of a molecule through the aperture.

This application is a continuation of co-pending U.S. application Ser.No. 11/399,663, which claims the benefit of U.S. Provisional ApplicationNo. 60/668,632, filed Apr. 6, 2005; U.S. Provisional Application No.60/688,799, filed Jun. 9, 2005; and U.S. Provisional Application No.60/727,603, filed Oct. 18, 2005, the entirety of all of which are herebyincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under National Instituteof Health grant “Electronic sequencing in nanopores” RO1HG003703. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates generally to detection and identification ofmolecules, and more particularly relates to molecular analysistechniques for characterization and sequencing of polymers, includingbiopolymers such as polynucleotides.

The detection, characterization, identification, and sequencing ofmolecules, including biomolecules, e.g., polynucleotides such as thebiopolymer nucleic acid molecules DNA, RNA, and peptide nucleic acid(PNA), as well as proteins, and other biological molecules, is animportant and expanding field of research. There is currently a greatneed for processes that can determine the hybridization state,configuration, monomer stacking, and sequence of polymer molecules in arapid, reliable, and inexpensive manner. Advances in polymer synthesisand fabrication and advances in biological development and medicine,particularly in the area of gene therapy, development of newpharmaceuticals, and matching of appropriate therapy to patient, are inlarge part dependent on such processes.

In one process for molecular analysis, it has been shown that moleculessuch as nucleic acids and proteins can be transported through a naturalor synthetic nano-scale pore, or nanopore, and that characteristics ofthe molecule, including its identification, its state of hybridization,its interaction with other molecules, its sequence, i.e., the linearorder of the monomers of which a polymer is composed, can be discernedby and during transport through the nanopore. Transport of a moleculethrough a nanopore can be accomplished by, e.g., electrophoresis, orother translocation mechanism.

If the dimensions of the nanopore are such that an extended nucleic acidmolecule occupies a substantial fraction of the nanopore'scross-sectional area during translocation, the polymer molecule can becharacterized by and during transport through the nanopore by at leasttwo mechanisms. In a first of these, the translocating moleculetransiently reduces or blocks the ionic current produced by applicationof a voltage between the two compartmentalized liquid ion-containingsolutions in contact with each end of nanopore. In a second of these,the translocating molecule transiently alters the electron current,including the tunneling electron current, produced by applying a biasbetween two closely spaced local probes that are located to produce ananoscale gap, either on apposed points on the perimeter of the nanoporeor at opposite ends of a very short nanopore. Given that during itspassage through the nanopore each nucleotide in the polymer produces acharacteristically distinct modulation of the ionic current or theelectron current, the resulting sequence of either the ionic or theelectron current modulations can reflect the characteristics of thetranslocating polymer molecule.

Ideally, these molecular analysis techniques, like others that have beenproposed, should enable molecular characterization with single monomerresolution. Unambiguous resolution of individual monomer characteristicsis critical for reliable applications such as biomolecular sequencingapplications. But this capability is difficult to achieve in practice,due to several aspects of molecular detection and analysis in general.

First, for any molecular orientation, the speed at which a molecule ischaracterized, e.g., the speed at which a sequence of nucleotides isdetected, may impact the production of a useful molecularcharacterization signal. The ability to discern changes in acharacterization signal or other indicator from one monomer to the nextmay be highly sensitive to the speed at which the nucleotides arecharacterized. For example, the speed at which a nucleotide istransported through a nanopore may impact the degree of ionic currentblockage or electron current modulation caused by that nucleotide, ormay exceed the bandwidth of the measurement instruments that can befabricated to detect the very small pico- or nanoampere currents typicalof ionic current measurements or tunneling current measurements in ananopore.

Second, the physical orientation of a given nucleotide as it ischaracterized may impact the detection of characteristics of thatnucleotide. This difficulty is particularly acute when modulations ofthe electron current between two closely spaced local probes, orientedto produce a nanoscale gap, are to be sensed. Such modulations inelectron current, including tunneling current, between two closelyspaced probes are known to be especially sensitive to atomic scalevariation in the distance between the two probes or to the preciseorientation of molecules between the two probes. Thus, e.g., eachalternative nucleotide orientation within a DNA strand may produce adifferent detection and characterization signal or other indicator, andsuch signals may be ambiguous for multiple nucleotides or multiplemolecular features. For example, the orientation of a nucleotide as itpasses through a nanopore having a location-specific limiting asperitymay alter the electronic current modulation caused by that nucleotide atthe location of the asperity. Various nucleotides and various molecularattributes may result in similar, or indistinguishable electron currentmodulations, depending on their orientation as they are transportedthrough a nanopore. These examples illustrate that, in general, thechallenges of speed control and nano-scale spatial orientation limit theability to achieve precise, high resolution molecular characterizationsuch as biopolymer sequencing.

SUMMARY OF THE INVENTION

The invention overcomes the speed control and orientation controldifficulties of prior molecular characterization techniques by providingcharacterization devices and characterization methods that orient themonomers of molecules translocating through a nanopore. In one examplemolecular characterization device of the invention, there is provided afirst reservoir containing a liquid solution including a molecule to becharacterized and a second reservoir for containing a liquid solutionincluding a molecule that has been characterized. A solid state supportstructure is provided including an aperture having a molecular entranceproviding a fluidic connection to the first reservoir and a molecularexit providing a fluidic connection to the second reservoir.

In the molecular characterization device there is provided one carbonnanotube having a longitudinal sidewall disposed as a molecularcontacting surface at the aperture. A voltage source is connected inseries with the carbon nanotube for electrically biasing the carbonnanotube, and an electrical current monitor is connected in series withthe carbon nanotube for monitoring changes in electrical current throughthe nanotube corresponding to translocation of a molecule through theaperture.

This molecular characterization device and the characterizationtechniques it enables allow for fast, reliable, repeatable, anduncomplicated characterization of a wide range of molecules andmolecular configurations. Other features and advantages of the inventionwill be apparent from the following description and accompanyingfigures, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic cross-sectional view of a first exampleembodiment of a molecular characterization device provided by theinvention, articulated with end-oriented carbon nanotube probes;

FIG. 1B is a schematic side view of a carbon nanotube, identifying theend and side of the nanotube;

FIG. 1C is a schematic cross-sectional view of the device of FIG. 1A,shown here orienting a ssDNA molecule as the molecule is characterized;

FIG. 2A is a schematic cross-sectional view of a further exampleembodiment of a molecular characterization device provided by theinvention, articulated with one end-oriented nanotube probe and oneside-oriented nanotube probe;

FIG. 2B is a schematic planar view of the embodiment of FIG. 2A;

FIG. 2C is a schematic cross-sectional view of the embodiment of FIG.2A, shown here orienting a ssDNA molecule as the molecule ischaracterized;

FIG. 3A is schematic cross-sectional view of a further exampleembodiment of a molecular characterization device provided by theinvention, articulated with end-oriented carbon nanotube probes and aside-oriented translocation control carbon nanotube;

FIG. 3B is a schematic planar view of the embodiment of FIG. 3A;

FIG. 3C is a schematic cross-sectional view of the embodiment of FIG.3A, shown here controlling translocation speed and orienting a ssDNAmolecule as the molecule is characterized;

FIG. 4A is schematic cross-sectional view of a further exampleembodiment of a molecular characterization device provided by theinvention, articulated with one end-oriented carbon nanotube probe, oneside-oriented nanotube probe, and a side-oriented translocation controlcarbon nanotube;

FIG. 4B is schematic cross-sectional view of a further exampleembodiment of a molecular characterization device provided by theinvention, articulated with one end-oriented carbon nanotube probe andone side-oriented nanotube probe extending through the length of ananopore.

FIG. 5A is a schematic cross-sectional view of a further exampleembodiment of a molecular characterization device provided by theinvention, articulated with end-oriented carbon nanotube probes, andconfigured for translocation and characterization of DNA-nanotubecomplexes through a nanopore;

FIG. 5B is a schematic cross-sectional view of a further exampleembodiment of a molecular characterization device provided by theinvention, articulated with end-oriented carbon nanotube probes, andconfigured for translocation of actuator tip-mounted DNA-nanotubecomplexes through a nanopore;

FIG. 5C is a schematic cross-sectional view of a further exampleembodiment of a molecular characterization device provided by theinvention, configured with an actuator tip-mounted carbon nanotube probefor scanning a DNA-nanotube complex;

FIG. 6 is a schematic cross-sectional view of a molecularcharacterization device provided by the invention, configured with ananopore in a silicon nitride membrane on a silicon support frame;

FIG. 7 is a schematic view of a packaged molecular characterizationdevice provided by the invention including fluid reservoirs andchannels;

FIGS. 8A-8F are schematic cross-sectional views showing fabricationsteps in an example process for producing a molecular characterizationdevice;

FIGS. 9A-9L are schematic cross-sectional views showing fabricationsteps in a further example process for producing a molecularcharacterization device;

FIGS. 10A-10L are schematic cross-sectional views showing fabricationsteps in a further example process for producing a molecularcharacterization device; and

FIGS. 11A-11M are schematic cross-sectional views showing fabricationsteps in a further example process for producing a molecularcharacterization device.

DETAILED DESCRIPTION OF THE INVENTION

Referring to FIG. 1A there is schematically shown a first exampleembodiment of a molecular characterization device 10 provided by theinvention. For clarity of discussion, device features illustrated inFIG. 1 are not shown to scale. As shown in FIG. 1A, in the device thereis provided a nano-scale aperture, or nanopore 12, in a supportstructure 14. On a first side of the support structure is a first liquidcompartment 16, or reservoir, containing a liquid solution includingmolecules to be characterized, and on an opposite side of the supportstructure is a second liquid compartment 18, or reservoir into whichcharacterized molecules are transported by translocation through thenanopore. A molecular entrance at the inlet to the aperture providesfluidic communication between the first reservoir 16 and the aperture,while a molecular exit at the outlet of the aperture provides fluidiccommunication between the aperture and the second reservoir 18.

The molecular characterization enabled by the analysis device of theinvention includes a wide range of analyses, including, e.g.,sequencing, hybridization detection, molecular interaction detection andanalysis, configuration detection, and other molecularcharacterizations. The molecules to be characterized can include, ingeneral, any molecule, including polymers and biomolecules such asproteins, nucleic acids such as the polynucleotides DNA and RNA, sugarpolymers, and other biomolecules. In one application, shown in thefigure, the molecules to be characterized comprise single-stranded DNAmolecules (ssDNA) 20 having a sequence of nucleoside bases 22 to becharacterized, for example, by determining the identity of the sequenceof bases along each ssDNA backbone. For clarity of discussion thissequencing example will be employed in the following description, butsuch is not the exclusive application of the molecular characterizationdevice of the invention. In addition, the sequencing operation describedbelow is not limited to the example of DNA; the polynucleotide RNA cansimilarly be characterized. The discussion below is therefore notintended to be limiting to a particular implementation, but providesdetails of one example in a range of embodiments for molecularcharacterization.

In operation of the molecular characterization device of FIG. 1A,application of a voltage bias between the two liquid compartments 16,18, labeled “−” and “+” in the figure, causes molecules, e.g., ssDNAmolecules, provided in the first compartment 16, to beelectrophoretically driven, one at a time, into and through the nanopore12 to the second compartment 18, because the DNA backbone is negativelycharged when in solution. This voltage bias can be imposed by, e.g., theprovision of silver chloride electrodes 17, 19 immersed in the solutionsof the two compartments 16, 18, respectively, with corresponding voltagesources 21, 23, for controlling the voltage of each solution. Anelectrolytic solution of elevated pH or with selected denaturants isemployed, as explained in detail below, to maintain the DNA strands inthe first compartment 16 in an unstructured, single-stranded form priorto transport through the nanopore.

At the location of the nanopore 12 in the support structure 14 there areprovided electrically-contacted probes 24, 26 for directlyelectronically characterizing the nucleosides in the translocating DNAmolecules by local electron transport measurement. The probes 24, 26serve as two nano-scale electrodes that abut the nanopore 12 at pointson the perimeter of the nanopore, e.g., at opposite points of thenanopore perimeter. The probes 24, 26 are connected in an externalelectrical circuit 28 with a voltage source 30 for imposing a selectedvoltage bias between the probes, across the nanopore 12. Given that theelectrolytic solution containing the molecules passes through thenanopore, between the probes, it is preferred that the support structurebe provided as an electrically insulating structure and that anelectrically insulating layer 15 or support structure region be providedto electrically insulate the probes except at the tips or very smalllocal region of the probes that abut the perimeter of nanopore 12, asshown in FIG. 1A. This condition of electrical insulation enables theapplication of a selected voltage bias between the probes.

Given that the nanopore is a nano-scale gap, application of a voltagebias between the probes 24, 26 causes electron transport across thenanopore between the two probes, to complete the probe circuit 28. Whena molecule such as a ssDNA nucleoside base 32 is positioned in thenanopore 12 between the probes 24, 26, the atomic structure of that baseinfluences the electron transport across the nanopore. An ammeter 34 orother electrical current measurement device is provided in the circuit28 for measuring the current associated with the nucleotide. In thismanner, molecules translocating through the nanoscale aperture can becharacterized; in this example, each of the nucleotides along the ssDNAmolecule can be distinctly characterized, e.g., identified.

For a nano-scale aperture such as the nanopore 12, the dominant electrontransport mechanism through an insulating DNA molecule, or othermolecule, and the local electrolytic liquid environment in the nanoporeis for many applications understood to be quantum mechanical electrontunneling. The term “tunneling” is here meant to refer to all types ofelectron transport across the nanopore, such as “hopping” fromelectronic state to electronic state, and other such transport, that ismodulated by the presence of a molecule in the nanopore. Such electrontransport is known to be very sensitive to local atomic structure and istherefore well-suited for distinctly characterizing molecules, e.g., foridentifying nucleoside bases. But other electron transport mechanismscan occur alternatively or in addition to electron tunneling across thenanopore, and can be exploited for molecular characterization, such as aDNA sequencing operation. For example, mechanisms associated withinduced charge effects, inelastic electron transport, or transport alonga section of the length of a DNA molecule backbone can also be employedand are understood to provide sufficient sensitivity for discriminatingbetween different nucleotide atomic structures as the nucleoside basesare transported through the nanopore.

Whatever electron transport mechanism takes place, the resultingcurrent, which is sensed by the external circuit 28, is modulated by theindividual nucleoside bases 22 as ssDNA molecules 20 translocate throughthe nanopore 12. This individual nucleotide characterization is enabledby the geometry of the nanopore, causing the nucleotides to pass instrictly sequential, single file order through the nanoscale aperturebetween the probes 24, 26. Measurement of the electron flow between theprobes identifies the nucleotides, much as the electron flow between ascanning tunneling microscope tip and a surface can identify the atomson the surface. Thus, the application of a voltage bias between theprobes makes it possible to dynamically sense the transverse electricalconductivity of a translocating molecule.

This transverse electrical conductivity can be sensed in accordance withthe invention through electron tunneling or other electron transportmechanism as described just above. The invention is not limited to aparticular electron transport mechanism. In the discussion to follow,the electron transport mechanism of electron tunneling will beconsidered, but the invention is not limited to such. Typical electroncurrents in tunneling microscopes are nanoamps. This relatively largeelectron current, and the attendant relatively large signal to noiseratio, can be produced with the external circuit 28 in which the probesare connected.

Turning now to specifics of the probes 24, 26, at least one of theprobes is provided as a fullerene structure such as a carbon nanotube.The term “fullerene structure” is herein meant to refer to any cage-likehollow molecular structure composed of ordered hexagonal and pentagonalgroups of carbon atoms. One of the probes can also be provided as ametal conductor, but for many applications, it can be preferred toimplement both probes 24 26 as fullerene structures such as carbonnanotubes. Carbon nanotubes are hollow tubes formed primarily ofhexagonal groups of carbon atoms. Single-walled carbon nanotubes (SWNT)are one dimensional tubes that consist of a single rolled-up sheet ofgraphite having a crystalline, hexagonal, fullerene atomic structure.Carbon nanotubes can be synthesized with diameters as small as 7Ångströms and lengths from sub-micron to millimeter. Carbon nanotubesare characterized by extreme mechanical and chemical robustness, and canbe selected to exhibit either the excellent electrical transportproperties of graphite or the band-gapped electronic structure of asemiconductor. Thus, the crystalline structure of carbon nanotubesprovides a well-defined, predetermined, ordered, and robust morphologythat can withstand the aqueous environment and high local electric fieldconditions of the nanopore; for many applications a metal conductor maynot be expected to likewise do so.

In addition, because the electrical characteristics of carbon nanotubesare very sensitive to atomic scale perturbations, nanotubes are thepreferred nanoscale probe for electronically differentiating between thefour different DNA nucleoside bases translocating through a nanopore.Specifically, the van der Waals interaction of a DNA or RNA nucleosidecoupled to a nanotube, as well as the electrical properties of a DNA orRNA nucleoside, can significantly affect the electronic properties ofthe nanotube by influencing the free charge carrier concentration andlocation of charge carrier energy levels, and correspondingconductivity, of the nanotube. Thus, the electronic properties of thenanotube-part of a coupled DNA base-nanotube hybrid play as important arole in determining the measurable contrast between analyzed DNA basesas do the molecules to be sequenced, the local environment near themolecule-nanotube hybrid, and the electrical conditions established byvoltages applied to the nanotube probes and the ssDNA electrolytesolution.

In the example nanotube probe configuration of FIG. 1A, the nanoporeprovides a stable electron transport gap between cut ends of a singlenanotube, or between the ends of two nanotubes, such that the nanotubeends can serve as electron source and sink for electron transport acrossthe nanopore. With this arrangement, nano-scale resolution of theindividual nucleoside base structures can be directly achieved and theirelectrical properties make it possible to detect and characterize manyaspects of the polynucleotide, including its sequence. Exampletechniques for synthesizing nanotubes and fabricating nanotube probearrangements are described in detail below.

The nanotube probes can be provided as metallic or semiconductingstructures, but for many applications a small-gap semiconductingnanotube probe can be preferred to enable the production of measurableelectron transport current at reasonable probe voltage biases. A singlewall nanotube probe structure can also be preferred. The inventioncontemplates a range of fullerene structures to be employed as nanoporeprobes. For example, bucky ball fullerene spheres embedded in anelectrically conducting medium can be employed as nanopore probes.Semiconducting nanowire structures can also be employed as nanoporeprobes. In the discussion to follow the example of nanotubes is employedbut such is not intended to be limiting.

It has been discovered in accordance with the invention that thenanotube probes 24, 26, can be employed to physically orient nucleosidebases as the bases translocate through the nanopore, while at the sametime producing a direct electronic signal that is indicative of thetranslocating bases, for characterizing the bases. This physicalorientation is achieved in accordance with the invention by exploitingthe affinity of DNA strands to closely couple with a surface of a carbonnanotube. When in the presence of a fullerene structure such as a carbonnanotube, each nucleoside base of a DNA molecule tends to formπ-stacking interactions with the nanotube fullerene structure, see forexample Zheng et al., Nature Materials 2, 338-342, May 2003. A DNAmolecule is understood to be quite flexible in bond torsion within thesugar-phosphate backbone, and it is this flexibility that accommodatesthe nucleoside base-nanotube coupling. In the coupling, the planar basesof a DNA molecule can individually be associated with, or coupled to, ananotube surface by way of a non-covalent absorption process, in acondition whereby the plane of a base lays flat against the nanotubesurface. As a result, in this configuration, a DNA nucleoside base isphysically aligned by a nanotube surface to an orientation correspondingto that of the nanotube surface.

This phenomena of DNA-nanotube coupling is understood to be driven byfree-energy considerations, electrical charge states, the complementaryhydrophobic/hydrophilic nature of DNA bases and carbon nanotubes, andDNA backbones, respectively, and in part by the van der Waalsinteraction between a nucleoside base and the surface structure of ananotube. No particular conditions need be imposed to enable thecoupling of a nucleoside base to a nanotube surface. Indeed, it has beensuggested that DNA or RNA will preferentially couple to nanotubesurfaces rather than aggregate with itself.

In accordance with the invention, in the example configuration of FIG.1A, a proximate nanotube probe is provided at a point along the lengthof the nanopore such that a DNA base will tend to align with a surfaceof the nanotube as the base is translocated through the nanopore. FIG.1B is a schematic view of a nanotube 24, identifying the variousnanotube surfaces available for coupling to a base and the terminologyused herein for those surfaces. The nanotube 24 includes a side 27 towhich a base can couple, and includes ends 29 and 31 to which a base cancouple. The ends can be provided as a continuous extension of thegraphitic surface, as in end 29, or can be a cut face end, as in end 31.

FIG. 1C is a schematic view of the molecular characterization device ofFIG. 1A, here shown at a time wherein a nanotube probe 24 is physicallyaligning a nucleoside base 32 as the base translocates through thenanopore. A nucleoside base 32 is coupled to an end of one 24 of thenanotube probes as the ssDNA 20 is translocated through the nanopore 12.As explained above, the planar nucleoside base 32 has a tendency to layflat against the nanotube. This configuration enables a preciseorientation of the base in the nanopore for detecting an electrontransport current that is distinctly representative of the base. Thecoupling of the base with the nanotube end depicted in FIG. 1C isintended to be schematic only and does not represent particular detailsof the base orientation relative to the nanotube 24.

To enable the translocation and coupling depicted in FIG. 1C, theelectrophoretic force across the nanopore is controlled to allow thenucleoside base 32 to slide along the nanotube end, with theelectrophoretic force being sufficiently strong to cause the base todecouple from the nanotube end after some amount of time. Thiselectrophoretic force control is implemented by control of the relativevoltage between the two liquid compartments 16, 18 from control of thevoltage sources 21, 23, respectively, in FIG. 1A and 1C.

The binding energy between ssDNA and a nanotube has been estimated to beabout −1.0 eV/nm, see, e.g., Zheng et al., “DNA-assisted dispersion andseparation of carbon nanotubes,” Nature Materials, 2, 338-342, 2003. Thecorresponding force needed to remove a nucleoside from an unbiasednanotube is therefore between about 3 pN and about 6 pN, which isapproximately the electrophoretic force applied to a ssDNA traversing ananopore having a voltage bias of between about 100 mV-200 mV across thenanopore, see, e.g., Sauer-Budge et al., “Unzipping kinetics ofdouble-stranded DNA in a nanopore,” Phys. Rev. Lett. 90,2381011-2381014, 2003. Hence, the application of a 200 mV bias acrossthe nanopore will, absent a bias on the nanotube probes, cause thenucleosides to slide across the nanotube end as shown in FIG. 1C. Thecoupling that occurs between a molecule and a carbon nanotube istherefore to be understood to be non-covalent, non-permanent, and of anature that enables a molecule to slide along a nanotube surface whilecoupled to the surface.

As shown in FIG. 1C, the nanotube 24 to which the base 32 is coupled ispositively biased with respect to the second nanotube 26. If the voltageof the nanotube 24 results in a positive bias of that nanotube relativeto the electrolytic solution in the nanopore, then the negativelycharged nucleotides will be prevented from sliding across the nanotubeuntil the positive bias of the nanotube 26 is briefly reduced or untilthe bias across the nanopore, between the two liquid compartments, israised sufficiently to slide the nucleotide across the nanotube. Theinvention therefore enables the control of translocation speed ofnucleotides as well as orientation of the nucleotides as theytranslocate through the nanopore.

In the example configurations of FIG. 1A and 1C the nanotube probes 24,26 are shown to be oriented with an end of the nanotube abutting theperimeter of the nanopore. It is understood that the end of a nanotubecan have a hexagonal carbon surface structure like that of the side of ananotube, or other more complex detailed geometrical characteristic, andtherefore can impose the same nucleotide orienting influencedemonstrated by the side of a nanotube. If in a given application orconfiguration it is found that an end of a nanotube does not provideadequate orienting influence, then the nanotube end can befunctionalized or otherwise processed, in the manner described below, toprovide a terminating surface having the requisite nucleotide orientingcharacteristics.

With this example, it is shown that the nanotube probes enable bothdirect molecular characterization as well as molecular orientationcontrol, and as described below, can further provide moleculartranslocation speed control. The nanotube probes are therefore not asimple alternative to conventional metallic probes and instead, based onthe discovery of the invention, can be employed for enabling precisecontrol of molecular characterization.

Referring to FIG. 2A, in a further embodiment provided by the invention,the molecular-orienting surface of one of the nanotube probes isprovided as the side of a nanotube rather than a nanotube end. As shownin FIG. 2A, a first nanotube probe 24 is provided in the mannerdescribed above with an end disposed at a point along the length of ananopore 12. The other nanotube probe 50 is disposed orthogonal to thefirst probe 24, so that a side of this probe 50 abuts the perimeter ofthe nanopore 12.

FIG. 2A is a cut-away sideview of the configuration, showing only thenanopore and the nanotube probes in a support structure 14, for clarity,but it is to be understood that liquid compartments, extended supportstructure, and electrical connections like that of FIG. 1A are included.In addition, as shown in FIG. 2A, the support structure and anelectrically insulating region 15 are provided for electricallyinsulating the two nanotubes except where they abut on the nanoporeperimeter.

The electrical connections for this configuration are shown in FIG. 2B,which is a schematic top-down, planar view of the assembly. Here isshown the nanopore 12 in a support structure 14, with an orthogonalpositioning of the two nanotube probes 24, 50, resulting in a T-shapedprobe arrangement. For clarity, the electrically insulating supportstructure and coating material that is located on top of the nanotubeprobes is not shown in FIG. 2B, enabling a direct view of the nanotubeprobe locations. With the arrangement of FIG. 2B, a first externalcircuit 28 is provided, in the manner described above with reference tothe embodiment of FIGS. 1A-1B, to provide for application of a selectedvoltage bias 30 between the nanotube probes 24, 50. This electrical biasimposes a selected voltage across the aperture of the nanopore forstimulating electron transport across the nanopore as nucleoside basesof a DNA molecule are transported through the nanopore in the mannerdescribed above.

Referring also to FIG. 2C, there is shown the condition in which anucleoside base 32 is coupled to the side of one 50 of the nanotubeprobes as the ssDNA 20 is translocated through the nanopore. Thisconfiguration enables a precise orientation of the base in the nanoporefor detecting an electron transport current that is distinctlyrepresentative of the base. The base can slide along the side of thenanotube 50 as the base is characterized. Note also in FIG. 2C, with thepolarity of the voltage source 30 as-shown in FIG. 2B, the side-orientednanotube probe 50 is biased electrically positive relative to theend-oriented nanotube probe 24. If this positive bias is also positivewith respect to the electrolytic solution, then as a result, as anucleoside base 32 is drawn through the nanopore 12, thenegatively-charged DNA backbone is electrically attracted to theside-oriented probe 50. The nucleoside base 32 interacts with, andcouples to, the side of the probe 50 and remains coupled until the biasis reversed.

As a result, the side-oriented nanotube probe 50 can be furthercontrolled for enabling control of nucleoside base translocation speedas a base is transported through the nanopore. Referring again to FIG.2B, ends 58, 60 of the side-oriented nanotube probe 50 are connected ina second external circuit 52 provided with a voltage source 54 forseparately controlling the voltage of the longitudinal nanotube probe50; an ammeter or other current control and measurement device 56 canalso be included in the second external circuit 52.

As explained above, the coupling of a DNA strand with a nanotube surfacecan at least in part be controlled by the relative electrical charge ofthe strand and the nanotube surface. Because a DNA backbone isinherently negatively charged when in solution, a positively chargednanotube surface, relative to the solution, tends to attract the DNAbackbone to the nanotube surface. Conversely, a negatively chargednanotube surface tends to repel the DNA backbone from the nanotubesurface. These conditions are exploited in accordance with the inventionto control the duration of the coupling of a nucleoside base with theside-oriented nanotube probe.

Referring again to FIG. 2C, when the side-oriented nanotube probe 50 iselectrically biased positively with respect to the electrolyticsolution, the DNA backbone tends to be attracted to the side-orientednanotube probe 50 and the nucleoside base 32 couples with theside-oriented nanotube probe 50. During this coupling, the electrontransport that occurs between the nanotube probes, across the nanoporethrough the DNA base, can be measured for identifying the base.

Once this base identification measurement is complete, the secondexternal circuit 52 is controlled to adjust the voltage source 54 andthe corresponding electrical bias of the side-oriented nanotube probe50. The voltage bias is now selected so that the side-oriented nanotubeprobe 50 is biased negatively with respect to the electrolytic solution,whereby the DNA backbone tends to be repelled from the side-orientednanotube probe 50. With the initiation of the resulting repelling force,the nucleoside base 32 de-couples from the side-oriented nanotube probe50, resulting in the configuration of FIG. 2A, and is transportedfurther down through the nanopore by the electrophoretic force. The nextsequential nucleoside base to be identified is correspondinglytransported to the location of the nanotube probes for the next baseidentification cycle.

By enabling this base identification cycling, the embodiment of FIGS.2A-2C imposes on a DNA strand both translocation speed control as wellas physical orientation control on nucleotides of the strand as thosenucleotides translocate through a nanopore. The example implementationof FIGS. 2A-2C is particularly advantageous in that the side-orientednanotube probe 50 provides nanotube ends 58, 60 away from the nanoporeand at which electrical connection to an external circuit 52 can bemade. But if necessary or desirable for a given implementation, theend-oriented nanotube probe 24 can instead be separately controlled withthe second external circuit 52 for causing controlled coupling andde-coupling of a DNA nucleotide to the end-oriented nanotube probe 24rather than the side-oriented nanotube probe 50.

As explained above, it is understood that a DNA nucleotide has atendency to couple with both the ends of a nanotube as well as the sidesof a nanotube. Whether a nanotube end or a side is employed for DNAcoupling, the invention does not require that all nucleoside bases beoriented identically by a nanotube surface. Instead, it is preferredthat all nucleoside bases of a common type be oriented substantiallysimilarly; i.e., all T bases should be oriented similarly, all G basesshould be oriented similarly, and so on. With this condition met, it canbe assured that each instance of a given base type will result in anexpected orientation or in a limited class of orientations, andtherefore will aid in contrasting between the four DNA bases andfacilitate precision of sequencing.

Further, the invention does not require a particular orientation forcoupling of a nucleoside base to a nanotube surface. It is understoodthat under many conditions, the plane of a nucleoside base tends to layflat against a nanotube surface, as explained above. But such is notuniversally required by the invention. A nucleoside base can be arrangedat some angle with a nanotube surface, can effectively stick off thesurface, or be oriented in any suitable manner that enables electronicdetection of its identity. As explained above, it is preferred that eachinstance of a given base type be oriented similarly, to enhance basecontrast precision, but no particular orientation is required.

In a further embodiment provided by the invention, moleculartranslocation control can be implemented separately from the electrontransport nanotube probe configuration. FIG. 3A is a schematiccross-sectional view, not to scale, of an example of such anarrangement. As in FIG. 2A and C above, it is to be understood that theliquid compartments and associated voltage sources are included as inFIG. 1A, and that electrical insulation of both nanotube probes isprovided, with only the ends of the nanotubes abutting the nanoporebeing un-insulated.

In FIG. 3A there are shown two nanotube probes 24, 26 in a supportstructure 14, in the manner of FIG. 1A, across a nanopore 12. Thenanotube probes are in this example arranged with an end-orientation atthe nanopore perimeter. At the support structure surface 63 at which aDNA strand 20 is to enter the nanopore 12 there is provided aside-positioned nanotube 65 at the perimeter of the nanopore 12. Theelectrical bias of this surface-positioned nanotube 65 is controlled inthe manner of the side-oriented nanotube probe 50, in FIGS. 2A-2C, forcontrolling the translocation speed of the DNA strand being transportedthrough the nanopore 12 for base identification at the location of thenanotube probes 24, 26.

FIG. 3B is a top down planar view of the configuration of FIG. 3A, hereincluding the external circuit connections for the configuration. Inthis view, the nanotube probes 24, 26 are indicated with dottedcross-hatching as underlying the surface of the support structure 14 ata point along the length of the nanopore 12. The surface-positionednanotube 65 is purely cross-hatched to indicate its position above thenanotube probes 24, 26, on the surface of the support structure 14. Afirst external circuit 28 like that of FIG. 1A is provided for the twonanotube probes 24, 26. A voltage source 30 in the circuit 28 enablesapplication of a selected voltage bias between the nanotube probes 24,26 for causing electron transport across the nanopore and enablingelectrical current measurement for nucleoside base identification, inthe manner previously explained.

A second external circuit 52, like that of FIG. 2B, is provided forelectrically biasing the surface-positioned nanotube 65. The ends 66, 68of the surface-positioned nanotube 65 are connected in the secondexternal circuit 52 provided with a voltage source 54 for controllingthe voltage of the surface-positioned nanotube 65; an ammeter or othercurrent control and measurement device 56 can also be included in thesecond external circuit 52.

Referring also to FIG. 3C, when the surface-positioned nanotube 65 iselectrically biased positively with respect to the electrolyticsolution, the DNA backbone tends to be attracted to thesurface-positioned nanotube 65 and a nucleoside base 32 couples with thesurface-positioned nanotube 65. This acts to control the forwardtranslocation speed of the DNA strand through the nanopore. The couplingof the base 32 to the surface-positioned nanotube 65 also spatiallyorients the base by orienting the base on the nanotube surface. At thesame time, the first external circuit 28 imposes a voltage bias betweenthe nanotube probes 24, 26. The resulting electron transport that occursbetween the nanotube probes, 24, 26, across the nanopore through the DNAbase located at the position of the nanotube probes 24, 26, can at thistime be measured for identifying the base at the position of thenanotube probes 24, 26.

Once this base identification measurement is complete, the secondexternal circuit 52 is controlled to adjust the voltage source 54 andthe corresponding electrical bias of the surface-positioned nanotube 65.The voltage bias is now selected so that the surface-positioned nanotube65 is biased negatively with respect to the electrolytic solution,whereby the DNA backbone tends to be repelled from thesurface-positioned nanotube 65. With the initiation of the resultingrepelling force, the nucleoside base 32 de-couples from thesurface-positioned nanotube 65, resulting in the configuration of FIG.3A, and is transported into the nanopore by the electrophoretic force.The next sequential nucleoside base to be identified is correspondinglytransported to the location of the nanotube probes for the next baseidentification cycle. The nucleoside base orientation that was carriedout at the surface-positioned nanotube 65 may remain with the base as itenters the nanopore and approaches the nanotube probes, depending on thelength of the nanopore. This orientation control is then reinforced bythe coupling of the base to one of the nanotube probes as the basearrives at the location of the probes along the nanopore length.

In an alternative implementation, the surface-positioned nanotube 65 canbe end-oriented at the perimeter of the nanopore 12 rather thanside-oriented at the nanopore perimeter. This end-orientation of thesurface-positioned nanotube disposes an end at the nanopore edge in themanner of the end-oriented nanotube probes 24, 26 described above. Thisimplementation may be less preferable, however, in that such does notprovide two nanotube ends at locations away from the nanopore perimeterfor making contact to the nanotube. If, however, a given applicationaccommodates contact to the end face-oriented surface of asurface-positioned nanotube, then such can be employed where applicable.

In a further embodiment of the invention, the first external circuit 28shown in the example implementations of FIGS. 1, and 3 can be controlledin the manner of the second external circuit 52 shown in FIGS. 2B and 3Bto transiently bias the end-oriented nanotube tunneling probes 24, 26(FIGS. 1 and 3). Transient nanotube probe biasing is here imposed toenable with only a single circuit the translocation control achieved bythe second external circuit 52 as in the example implementation of FIG.2B. It is understood in accordance with the invention, and as explainedabove, that the electrical charge of the two nanotube probes causes thenegatively charged DNA backbone to be attracted to that nanotube probewhich is biased most positively with respect to the electrolyticsolution. When a voltage bias is applied between the nanotube probes,the DNA backbone is attracted to the positively charged nanotube probe,and the nucleoside base at that position couples with the positivelycharged nanotube probe. This coupling acts to slow or halt theprogression of the strand through the nanopore. When the voltage bias isreversed with respect to the electrolytic solution, the DNA backbone isrepelled from the nanotube to which the base was coupled, and the DNAstrand can then proceed through the nanopore. Control of the DNA strandtranslocation is thusly achieved.

Use of this transient nanotube probe scenario with the implementation ofFIG. 1 enables the two nanotube probes 24, 26 to conduct both spatialmolecular orientation control as well as molecular translocation speedcontrol. In the implementation of FIG. 3 this transient nanotube probebias control enables dual translocation speed controls imposed by thesurface-positioned nanotube 66 and the nanotube probes 24, 26. The twotranslocation controls here can be operated synchronously to cause aratchet-like translocation of the DNA strand through the nanopore.

In accordance with the invention, the various features and controltechniques incorporated into the example configurations of FIGS. 1-3 canbe selected as-desired for a given molecular characterizationapplication in a wide range of alternative arrangements. For example, asshown in the cross-sectional cut-away view of FIG. 4A, nanotubetunneling probes can be provided as an end-oriented probe 24 and aside-oriented nanotube probe 50. A surface-positioned nanotube 65 canfurther be provided for imposing additional molecular translocationspeed control. All of the nanotubes are electrically insulated, exceptfor the end or side of the nanotube abutting the nanopore. As shown inFIG. 4B, nanotube probes can further be provided as, e.g., anend-oriented probe 24 and a side-oriented nanotube probe 67 that extendshorizontally through the thickness of the support structure, along theinside length of the nanopore. With this arrangement, a nucleoside basecan slide along the side-oriented nanotube 67 through the full nanoporelength in a well-controlled orientation. To enable this condition, theside-oriented probe here is not insulated along the region of the probeexposed in the nanopore, as explained in the examples below. As can berecognized, the end-oriented probe 24 can also be provided as aside-oriented probe like the side-oriented probe 50 shown in FIG. 4A.

Each of these device arrangements can be controlled by one or moreselected external biasing circuits for imposing time-dependentorientation and translocation speed control. For example, a first biascircuit like the external circuit 28 in FIG. 1A can be employed betweenthe nanotube probes 24, 50, and a second bias circuit like the externalcircuit of FIG. 2B can be employed to separately control the bias of thelongitudinal nanotube probe 50. A further control circuit like theexternal circuit 52 in FIG. 3B can further be employed for controllingthe bias of the surface-oriented nanotube 65.

The example device configurations discussed above have been described asoperating by measuring electron transport through a molecule in ananopore, between nanotube probes. The invention does not limit thedevice configurations to such operation, however. It is also recognizedthat the proximity of a molecule to a nanotube can change theconductance of the nanotube along the nanotube length, by inducingchanges in the concentration of nanotube electronic carriers availablefor electronic transport along the length of the nanotube, as describedabove, and/or by modulation of the electrical mobility of suchelectronic carriers. Therefore, all of the device configurations thataccommodate interaction of a molecule with a side of a carbon nanotubecan also be operated in what can be termed an “FET mode.” Examples ofsuch configurations are those shown in FIGS. 2A-C (nanotube probe 50),FIGS. 3A-3C (nanotube 65), and FIG. 4A (nanotubes 50 and 65).

In the FET mode of operation, an electrical bias voltage is appliedacross opposite ends of a nanotube having a side that forms themolecular contacting and orienting surface, e.g., nanotubes 50 and 65 inFIGS. 2-4. This bias can be applied with the circuit 52 in FIGS. 2B and3B, connected to nanotubes 50 and 65. The conductance of these nanotubesis sensitive to the proximity and characteristics of a molecule in thenanopore. Thus, measurement, in the circuit 52, of the electricalcurrent through the nanotube 50 or 65 as a molecule translocates throughthe nanopore provides an alternative technique for electronicallycharacterizing a molecule with a side-oriented nanotube-basedcharacterization device. The invention thus is not limited to aparticular mode of operation of the molecular characterization devicesof the invention.

In addition, in all of the implementations of FIGS. 1-4, the voltagebias of each of the solutions in the two liquid compartments can beadjusted in a time-dependent manner to control the electrophoretic forcedriving a molecule through the nanopore relative to themolecule-nanotube coupling force, as explained above. For example, theelectrodes 17, 19 in the first liquid compartment 16 and second liquidcompartment 18, respectively (FIG. 1A), can be selectively biased overtime, e.g., in synchrony with applied nanotube probe bias control andtranslocation speed bias control. Such liquid bias provides a furtherlevel of control over the bias of the nanotube probes relative to theelectrolytic solution, and thus enhances the precision of moleculartranslocation control. Such precision can be desirable to achieve atranslocation speed that is commensurate with the sensitivity andbandwidth of the measurement electronics. At a translocation speed of,e.g., about 10⁴ bases/sec, measurements of distinct electron transportsignals for sequential bases can be resolved. Thus, with electrical biascontrol applied to a selected arrangement of nanotubes and externalcircuits, there can be achieved control of the rate of DNA translocationand the physical orientation of the DNA nucleoside bases in a mannerthat accommodates direct electronic discrimination between the differentnucleoside bases.

As a result of these capabilities, the molecular analysis device of theinvention functions as a very high throughput sequencing device, eventhough the nanopore operates as a single molecule detector. Thousands ofdifferent molecules or thousands of identical molecules can be probed ina few minutes with the molecular analysis device of the invention.Because the method of operation of the nanopore device directly convertscharacteristic features of a translocating molecule into an electricalsignal, transduction and recognition can occur in real time, on amolecule-by-molecule basis. Further, long lengths of DNA can be probedin this real time fashion. While practical considerations of samplepreparation or of a particular application may limit the length of a DNAstrand that can be analyzed as the strand translocates through ananopore, there are no theoretical limits to analyzed strand length.

Turning now to further embodiments of the molecular analysis device ofthe invention, and referring to FIG. 5A, in a further molecular analysistechnique of the invention, DNA sequencing is conducted bytranslocation, through a nanotube-articulated nanopore, of complexes ofDNA strands coupled to free nanotubes. In a device provided by theinvention for conducting this process, as shown in FIG. 5A, in a firstliquid compartment 16 there are provided, in solution, ssDNA molecules100 which have been coupled to nanotubes 102, with at least one DNAstrand 100 coupled to separate nanotubes 102. Such a solution ofDNA-nanotube complexes can be formed and processed by, e.g., gelelectrophoresis, to provide desired molecule-nanotube complexes astaught in U.S. Patent Application Publication No. 2005/0009039, Jagotaet al., “Dispersion of Carbon Nanotubes by Nucleic Acids,” the entiretyof which is hereby incorporated by reference.

The first liquid compartment is in communication with a nanopore 12provided in a support structure 14 in the manner described above. At apoint along the length of the nanopore are disposed nanotube probes 24,26. The nanotube probes can be provided as end-oriented probes, in themanner shown in FIG. 5A, or can be side-oriented, e.g., in the mannershown in FIG. 2A.

Application of a voltage bias between the two liquid compartments 16,18, labeled “−” and “+” in the figure, causes the DNA-nanotube complexesin the first compartment 16 to be electrophoretically driven, one at atime, into and through the nanopore 12 to the second compartment 18.This voltage bias can be imposed by, e.g., the provision of silverchloride electrodes 17, 19 immersed in the solutions of the twocompartments 16, 18, respectively, with corresponding voltage sources21, 23, for controlling the voltage of each solution.

A voltage bias is applied between the nanotube probes 24, 26, across thenanopore, by an external circuit 28 having a controllable voltage source30. When a DNA-nanotube complex 104 is translocated into the nanoporeand translocates between the nanotube probes 24, 26, electron transportbetween the nanotube probes 24, 26 is influenced by the presence of theDNA-nanotube complex 104. An ammeter 34 or other current measuringelement is provided in the external circuit to monitor the resultingcurrent for identifying the bases of the DNA-nanotube complex. Giventhat each nucleoside base of a DNA molecule is coupled to thelongitudinal sidewall of a nanotube in a DNA-nanotube complex, then asthe DNA-nanotube complex translocates through the nanopore 12, eachnucleoside base of the coupled DNA strand is separately and distinctlyidentified by changes in the electron transport between the nanotubeprobes. It is to be recognized that the coupling of the DNA molecule tothe carbon nanotube can result in a reduction in speed of moleculartransport through the nanopore, relative to an un-coupled DNA strand,due to the added viscous drag of fluid near the nanopore caused by thepresence of nanotubes. But fluctuations in DNA strand movement due tobrownian motion are reduced by the coupling of the DNA strand to ananotube, due to the large mass and rigidity of the nanotube.

In addition to this electronic analysis of a translocating DNA-nanotubecomplex, changes in the ionic current flowing in solution through thenanopore from the first liquid compartment 16 to the second liquidcompartment 18, in the manner described previously, can be monitored toascertain the location of a translocating DNA-nanotube complex. Giventhat the dimensions of the nanopore correspond to that of theDNA-nanotube complex so that a complex occupies a large fraction of thenanopore's cross-sectional area during translocation, the complextransiently reduces, or blocks, the ionic current resulting from thevoltages applied to the first and second liquid compartments in themanner described above. As a result, measurements of the ionic currentbetween the first and second liquid compartments can be employed toindicate if a DNA-nanotube complex is within the nanopore. Such anindication can be synchronized with control of the external circuit forinitiating measurement of electron transport current once presence of aDNA-nanotube complex in the nanopore is confirmed.

The slowing and orientation effects of the DNA carbon nanotube hybridcomplex can be used to advantage for molecular characterization witheither ionic current flow or electron current flow molecularcharacterization devices. The invention contemplates thecharacterization of a DNA-nanotube complex by either electron currentflow, as in the nanotube probe configurations described above, or in anionic current flow measurement technique. The measurement of the ioniccurrent between the first and second liquid compartments can be employedto indicate a range of features of a DNA molecule that is complexed witha nanotube. For example, complementary base-paired sequences of monomersthat cause “hairpin” loops to be formed within a polynucleotide strandare distinctly oriented on the nanotube, and hence produce modulationsof the ionic current that are different than that of non-complementarysequences. Thus, the DNA-nanotube complex can be characterized in asuitable manner, e.g., with ionic current flow monitoring, withoutinclusion of detection of electron current flow modulation.

In the example embodiment of FIG. 5A, each free nanotube 102 providesphysical orientation of the nucleotides of the DNA strand 100 coupled tothat nanotube. When a DNA-nanotube complex 104 translocates through thenanopore 12, additional orientation control can be imposed by thenanotube probes 24, 26 in the manner described above. For example, thevoltage bias between the nanotube probes 24, 26 can be controlled toattract the negatively-charged DNA backbone, and the nanotube to whichit is coupled, to the positively charged nanotube probe 24. This enablesspatial orientation control of the DNA-nanotube complex within thenanopore between the nanotube probes.

If desired for a given application, the various nanotube configurationsin the examples of FIGS. 1-4 can be implemented to control theorientation as well as translocation speed of a DNA-nanotube complex asthe complex is translocated through the nanopore. In addition, asexplained above, the electrophoretic force between the two liquidcompartments can be controlled by adjusting the voltage bias of the twoliquid compartments to further control the DNA-nanotube complextranslocation speed.

Turning to FIG. 5B, in a further embodiment of the invention, thetranslocation of a DNA-nanotube complex through a nanopore can bestrictly controlled by an externally-commanded actuating element. Inthis arrangement provided by the invention, a DNA-nanotube complex 104to be analyzed is provided mounted on a tip 108 of a cantilever 110 orother moveable element that can be actuated by a controlling mechanism112. In one example embodiment, the mounting tip 108 is located at theend of a cantilever 110 of an atomic force microscope (AFM). Anysuitable actuated and moveable tip structure can be employed as themounting tip. Scanning tunneling microscope tips, microelectromechanical(MEMs) structures such as actuated micromachined cantilever beams andbridges, and other system apparatus or custom-fabricated mechanicalarrangements can be employed for the DNA-nanotube complex mounting tip.

Whatever tip configuration is employed, the tip-mounted DNA-nanotubecomplex is translocated through a nanopore 12 in a support structure 14,moving between two nanotube probes 24, 26. With precise control of thetip 108 and cantilever 110, the tip-mounted DNA-nanotube complex can beslowly advanced, e.g., at a translocation speed as low as approximately1 nucleoside base/second, through the nanopore 12. At the location ofthe nanotube probes 24, 26, electron transport across the nanopore,through the DNA-nanotube complex, is influenced by the presence of theDNA-nanotube complex. In the manner described above, an external circuitcan be employed to measure the electron transport for discriminatingbetween the nucleoside bases of the DNA molecule in the DNA-nanotubecomplex. For applications in which external circuit measurementbandwidth may be limited, this arrangement can be advantageous forprecisely controlling translocation speed in a manner that accommodatesthe circuit bandwidth.

It is to be understood that the external bias and measurement circuit 28of FIG. 5A, while not shown in FIG. 5B for clarity of discussion is tobe included in the configuration of 5B for enabling nanotube probe biasand electron transport measurement. Further, for many applications, theliquid compartments 16, 18 of FIG. 5A are preferably further included inthis configuration to provide a medium in which the DNA-nanotubemolecules can easily translocate through the nanopore, and to enableadditional bias control.

In a further control technique, an additional voltage source 114 canalso be provided for electrically biasing the DNA-nanotube complex 104translocating through the nanopore. Like biasing the gate or base of aconventional transistor, bias of the DNA-nanotube complex influenceselectron transport between the nanotube probes. The voltage applied tothe DNA-nanotube complex changes the spatial intimacy of the DNAcoupling to the nanotube and correspondingly adjusts the energy levelsof the DNA-nanotube complex that are available to electrons transportedbetween the nanotube probes. The voltage applied to the DNA-nanotubecomplex can therefore be controllably varied to adjust and enhance theresolution and contrast of electron transport measurements fornucleoside base identification. The electrostatic potential of theliquid solution in which the DNA-nanotube complex is translocated canalso be adjusted, in the manner described above, for further tuning ofthe electron transport.

With a voltage applied to the DNA-nanotube complex, the nanotube of thecomplex can itself be employed as an electron transport probe in themanner of the nanotube probes 24, 26. In this scenario, one of thenanotube probes 24, 26 can be eliminated so that the electron transportfor nucleotide analysis occurs between one nanotube probe, e.g., probe24, and the nanotube of the DNA-nanotube complex 104. The externalcircuit for measuring current flow, like the circuit 28 of FIG. 5A, isin this case connected between the one nanotube probe 24 and thecantilever actuator 112.

The invention provides an experimental configuration for verifying thenature and characteristics of DNA-nanotube complexes to be analyzed bythe devices of FIGS. 5A-5B. One example implementation of thisexperimental configuration is shown in FIG. 5C. As shown in FIG. 5C,there is provided a DNA-nanotube complex 104 suspended between twoelectrically contacted electrodes 120, 122. The electrodes 120, 122 canbe provided as any suitable conductor, e.g., as metallic electrodes oras nanotubes. The electrodes 120, 122 are connected in a circuit with avoltage source 124 for electrically biasing the suspended DNA-nanotubecomplex 104.

An electrically-contacted probing nanotube 116 is provided mounted onthe tip 108 of a cantilever 110, e.g., provided by an AFM 112. A voltagesource 114 is provided for electrically biasing the tip-mounted probingnanotube 116. With this configuration, the AFM is first operated in thetraditional AFM mode to enable the tip-mounted probing nanotube 116 tolocate the suspended DNA-nanotube complex 104. Then the AFM is operatedin scanning tunneling microscopy mode (STM) to record electronic imagesof the DNA-nanotube complex as the probing nanotube 116 is scanned alongthe suspended complex 104. The resulting images geometrically revealeach base along the backbone of the coupled DNA strand.

If necessary, scanning of the suspended DNA-nanotube complex can beconducted with the complex in a dry state. In this case, it is preferredthat the complex be dried from, e.g., a solution including volatilesalts such as ammonium acetate and an amount of methanol sufficient toreduce the surface tension below that which would disrupt the complexstructure during the drying process. For many applications it can bepreferred to conduct the scanning in a liquid environment that simulatesthe liquid solutions employed in the sequencing configurations of FIGS.5A-5B.

Whatever the scan environment, as the scan proceeds, the tunneling biasvoltage of the probing nanotube 116 can be adjusted upon arrival at eachnucleoside base along the DNA strand to explore the electron transportcharacteristics of that base. This information is then employed inaccordance with the invention to optimize the bias to be applied to thenanotube probes in the configurations of FIGS. 5A-5B for enhancingcontrast between the four different DNA bases.

The example molecular characterization devices described above are ingeneral configured in a solid state support structure, e.g., a structureformed of a microelectronic material, for example, employing thematerials and arrangements taught in U.S. Pat. No. 6,627,067, Branton etal., “Molecular and Atomic Scale Evaluation of Biopolymers,” theentirety of which is hereby incorporated by reference.

Turning to particular aspects of the molecular characterization devicefabrication features, the solid state support structure in which ananopore is provided is in general formed of a microelectronic material,e.g., a silicon-based material such as single crystal silicon, siliconnitride, or polysilicon. In one particularly well-suited configuration,the support structure in which a nanopore is provided is formed of amembrane on a support structure, e.g., a silicon nitride membrane on asilicon support frame. This configuration is shown in FIG. 6. Here isprovided a suspended silicon nitride membrane 160 on a silicon supportframe 162, fabricated in the conventional manner. Silicon nitride is aparticularly well-suited material because of its generally electricallyinsulating property and resistance to degradation by a wide range ofliquids. A range of other materials can be employed for the membrane inwhich a nanopore is provided, however; the membrane material is ingeneral preferably chemically inert and/or resistant. Exemplarymaterials include silica, alumina, plastics, polymers, elastomers,glasses, or other suitable material.

Carbon nanotubes 24, 26 are provided on the membrane 160, in the mannerdescribed below, with a nanopore at the center of the membrane. Allsurfaces of the device in contact with the electrolytic molecule-bearingsolution are electrically insulated, except for the surfaces of thenanotubes that abut the nanopore perimeter. The silicon nitridemembrane, being electrically insulating, inherently provides forinsulating nanopore walls. The nanotubes 24, 26 and silicon supportframe 162 are preferably coated with a layer of electrically insulatingmaterial 15, e.g., alumina, hafnium oxide, or other selected insulatingmaterial.

The discussion below provides details of formation of an aperture thatcan be sized as a nanopore in a membrane and support structure. Ingeneral, the nanopore is sized for interaction with a molecule ofinterest; that is, the nanopore is of a diameter that is similar to theatomic width of a molecule of interest. For applications in which, e.g.,a single-stranded polynucleotide is to be translocated through thenanopore, a nanopore diameter in the range of about 1 nm-20 nm can bepreferred. No specific constraints are placed on the nanopore geometryother than that it be adequate to permit only a single polymer moleculeat a time to traverse the nanopore, and that the molecule travel in anextended conformation, e.g., without secondary structure. A generallycircular nanopore geometry can be preferred but is not required,non-circular nanopore profiles can also be employed.

Further, there is no particular length requirement of the nanopore; ananopore length of, e.g., about 0.1 nm-800 nm can be employed andproduced by processing of, e.g., a silicon nitride membrane. Asexplained above, in order to accommodate electron transport across thenanopore, the walls of the nanopore are electrically insulating. If themembrane is itself an insulating material, then no additional nanoporewall processing is required, aside from the considerations discussedbelow. If the membrane is electrically conducting, the walls of thenanopore can be coated with a selected insulating layer in the mannerdescribed below.

FIG. 7 is a schematic view of an example housing configuration 130 forpackaging a microfabricated molecular characterization device systemlike that of FIG. 6, and for connecting such to the requisite circuitry.As shown in FIG. 7, the example housing configuration includes liquidreservoirs 132, 134, along with liquid channels 136, 138, in, e.g., asilicone rubber (PDMS) chip holder 137, to implement liquid compartmentsfor the molecule-bearing electrolytic fluid to be translocated through ananopore. A microfabricated structure, e.g., a silicon frame 140, isprovided for supporting, e.g., a membrane in which a nanopore 142 isprovided. Nanotube probe contact pad connections 150, 152 are providedfor making electrical contact between an external bias and measurementcircuit 154 and nanotube probes. Finally, sealed connections 156 areprovided for enabling the application of an external circuit 158 forelectrically biasing the two liquid compartments without leakage of theliquids out of their compartments.

With this arrangement, it can be preferred that the contact area ofliquid to the cis, i.e., top, side of the support structure should be assmall as possible, e.g., less than about 1,000 μm², to minimize thecapacitance of the system. The channels 136 for delivery of liquid tothe cis side of the nanopore are preferably configured for delivery towithin less than about 300 μm of the nanopore. The channels preferablyare less than about 200 μm in diameter, and are connected to tubing thatis preferably less than about 200 μm in diameter, in order to minimizethe sample volume needed to fill the liquid compartment without airbubbles.

Turning now to more specifics of processes for producing the molecularcharacterization devices of the invention, a nanopore can be formed in asupport structure in any suitable method. Where the support structure isprovided as a membrane, the membrane is formed in the conventionalmanner, e.g., as taught in U.S. Pat. No. 6,627,067, incorporated byreference above. Then an initial aperture is formed in the membrane, forsubsequent processing of the membrane to produce a final, smallernanopore diameter. In one technique in accordance with the invention, aninitial aperture is formed in a membrane by, e.g., ion beam milling,electron beam etching, plasma etching, wet etching, or other selectedtechnique for forming an aperture through the thickness of the membrane.Then the initial aperture diameter is reduced through a selectedprocess. In one example technique, the aperture diameter is reduced tothat of a nanopore through a process of ion beam sculpting, as taught inU.S. Patent Application Publication US2005/0006224, Golovchenko et al.,“Pulsed Ion Beam Control of Solid State Features,” the entirety of whichis hereby incorporated by reference.

In the process of forming a nanopore, the surface of the nanopore, alongthe nanopore length, may be processed to minimize or inhibit adsorptionof ssDNA on the nanopore surface as the DNA translocates through thenanopore. It is known that various biomolecules, such as ssDNA, willtend to bind on either hydrophilic and cationic surfaces, via theanionic backbone of the strand, or will tend to bind on hydrophobicsurfaces via the nucleotides. It can therefore be preferred in theprocess of fabricating the nanotube-articulated nanopore configurationto either provide the support structure, in which the nanopore isformed, of a material that is inert to a molecule of interest, e.g.,ssDNA, or to coat the nanopore and support structure with a materiallayer that is inert to the molecule of interest. Thus, it can bepreferred to provide an insulating surface layer on the nanopore thatwill have a neutral surface charge when in contact with themolecule-bearing electrolytic solution. If the molecule-bearing solutionis at or near pH=7, aluminum oxide is an ideal coating material. If themolecule-bearing solution is pH≧9, hafnium oxide can instead bepreferred, as it indefinitely withstands a high pH environment withoutdegradation.

Where the nanopore is to be coated with a selected material layer, theprocess of atomic layer deposition (ALD) can be preferred as adeposition technique. ALD is particularly advantageous in conjunctionwith nanopore fabrication because it can modify the electrical charge aswell as material properties of a surface while yielding highly conformalstep coverage of many different materials, even over high-aspect-ratiostructures, with precise, single-Ångström thickness control. In additionthe ALD process can be used for controlling the pore size and forinsulating the nanotube electrodes away from their molecular sensingregions, as described in detail below.

Where aluminum oxide is the selected coating layer material, such can bedeposited by, e.g., atomic layer deposition (ALD) in a manner thatenables monolayer deposition control, e.g., as taught in U.S. PatentApplication Publication No. US2005/0241933, Branton et al., “MaterialDeposition Techniques for Control of Solid State Aperture SurfaceProperties,” the entirety of which is hereby incorporated by reference.

In an example process for producing a film by ALD on a nanopore, in afirst process step, the surfaces of the structure on which the film isto be deposited are prepared to react with a selected molecularprecursor or precursors. For many applications, a metal precursor isemployed for the ALD process, e.g., an ALD metal precursor can beprovided as ML_(x), where M=Al, Ha, Mg, W, Ta, Si, or other metal, andL=CH₃, Cl, F, C₄H₉, or other atomic or molecular ligand that produces avolatile molecule. For example, for deposition of an Al₂O₃ layer on ananopore the structure is first exposed to, e.g., UV/ozone, immediatelyprior to ALD deposition, in order to generate hydroxylated surfaces thatare highly reactive to a metal precursor for forming the alumina layer.

Once hydroxyl groups are produced on the structure surface, a precursor,e.g., the metal precursor trimethylaluminum [Al(CH₃)₃] (TMA) fordeposition of alumina, can be employed to react with the producedhydroxyls at the surface, i.e., a gaseous precursor including Al(CH₃)₃molecules is provided to react with the surface —OH sites. This reactionproduces volatile CH₄ gas molecules and forms Al(OH)₂,(CH₃) orAl(OH)(CH₃)₂ at the initial —OH sites. For such an alumina formationprocess a suitable reaction zone temperature is between about 200° C.and about 300° C.

The Al(CH₃)₃ reaction with the hydroxyl groups is self-terminating inthat during the reaction the initial surface ligands, —OH, are consumedand the surface becomes populated with L ligands that cannot furtherreact with the metal precursor. This self-limiting reaction process canresult in deposition of less than or more than a monolayer of materialon the structure and pore surfaces. In a next ALD process step, anyremaining unreacted Al(CH₃)₃ precursor and the produced CH₄ gas isflushed from the reaction chamber, e.g., with dry nitrogen or othersuitable carrier gas. In a next process step, water vapor is admittedinto the reaction chamber to cause the surface of the deposited layer tobe reactive with the selected precursor. In the current aluminadeposition example, the water vapor is provided to react with exposedCH₃ groups. The reaction of water vapor with available CH₃ groupsliberates CH₄ gas and attaches —OH groups at each —CH₃ site, resultingin a newly hydroxylated surface. This hydroxylation process selfterminates when all —CH₃ sites have reacted with the water vapor. Duringthis process, adjacent —Al(OH)₂ sites cross-link to produce watermolecules and a linked Al—O—Al network.

In a next process step, the remaining water vapor and the liberated CH₄groups are exhausted, e.g., with a nitrogen flushing step in the mannerdescribed above. This completes one ALD reaction cycle, producing alayer of alumina on the structure and pore surfaces and sidewalls. Thedeposited layer can then react with a next cycle of water vapor followedby Al(CH₃)₃ admitted into the reaction chamber for reaction. Theduration of each process step in one ALD reaction cycle is selected toenable sufficient reaction time without providing excessive precursor orexhaust. For example, in the example alumina deposition process, one ALDcycle can be employed as 1 s flow of the metal precursor vapor into thereaction chamber followed by 5 s nitrogen purge and then 1 s flow ofwater vapor followed by another 5 s nitrogen purge. A layer of alumina,which can be, e.g., about ⅓ of a monolayer thick, is formed after eachsuch cycle.

With these process conditions for ALD of an alumina layer, it wasexperimentally determined that the deposition rate of Al₂O₃ is 0.99±0.12Å per reaction cycle, independent of the total number of cycles. Thisdeposition rate was verified over 20-500 cycles. Thus, starting with a 2nm-diameter nanopore, the nanopore diameter can be reduced to 1 nm by 5cycles of the Al₂O₃ deposition process with an error of only about ±1.2Å.

In an alternative technique provided by the invention, a Teflon-likelayer can be deposited on the surface of the nanopore and the supportstructure, e.g., as taught in U.S. Pat. No. 5,888,591, Gleason et al.,“Chemical Vapor Deposition of Fluorocarbon Polymer Thin Films,” herebyincorporated by reference.

To articulate solid state nanopores with nanotube probes, it can bepreferred to integrate nanotubes with the nanopore in a selected supportstructure. In one fabrication technique, carbon nanotubes aresynthesized directly on a support structure of interest, as taught inU.S. Patent Application Publication No. 2005/0007002, Golovchenko etal., “Carbon Nanotube Device Fabrication,” the entirety of which ishereby incorporated by reference.

In this technique, precise control of catalyst properties, andcorrespondingly precise control of nanotube growth are enabled, suchthat single-walled nanotubes oriented horizontally, parallel to asupport surface, are selectively synthesized for configuring thenanotube probes. In this process, the catalyst layer is formed by vapordeposition of a solid catalyst material, by sputtering, molecular beamepitaxy, sol gel formation, E-beam evaporation, thermal evaporation, orother selected vapor deposition process on the support structure.Whatever vapor deposition process is selected, it preferably iscontrolled to enable very low coverage of the vapor-deposited film, suchthat no more than several monolayers of the selected catalyst materialare deposited on the membrane or support substrate.

In one example vapor deposition process, thermal evaporation of Fe usinga tungsten boat spot welded with Fe foil can be carried out under vacuumconditions, e.g., at a pressure of about 10⁻⁵ or 10⁻⁶ Torr, to produce aFe catalyst layer of selected thickness. Whatever catalyst material andvapor deposition process is employed, it is preferred that the resultingcatalyst layer thickness be less than about 2 nm, or considered anotherway, it is preferred that the catalyst layer be characterized by a layercoverage of about 8×10¹⁵ atoms/cm² or less. It is understood that as thecatalyst layer thickness is increased, the diameter of nanotubes thatare horizontally synthesized from the catalyst layer correspondinglyincreases, and above a threshold catalyst layer thickness, multi-walled,rather than single-walled, horizontal nanotubes are formed. A thincatalyst layer, e.g., of 2 nm in thickness or less, is understood to beadequate for predictably and reliably forming single-walled nanotubes.

In one example fabrication sequence, contact pads are first formed onthe selected support structure in the conventional manner, e.g., as alayer of palladium for forming an ohmic contact with the nanotubeprobes. The contact pads are formed near to where the nanopore is to belocated. A nanotube catalyst layer is then formed on top of the metalcontact pad layer or layers. Then using conventional lift-offtechniques, a patterned photoresist layer is removed, resulting inpatterned catalyst/electrode regions. This technique can be particularlyadvantageous because it enables patterning of both nanotube probecontact pads and catalyst layers in a single step. For applications inwhich it is acceptable for the extent of catalyst regions to coincidewith that of nanotube probe contact pads, this process can therefore bepreferred.

If for a given application, it is preferred that the catalyst regions donot fully cover the contact pads, then an additional lithographic andetch sequence can be carried out to remove catalyst material fromportions of the contact layer. In one example process, the catalystlayer is masked with, e.g., a patterned photoresist layer, exposingregions of the catalyst layer that are to be removed. A dry etchprocess, e.g., plasma etching, ion beam etching, or other technique, isthen employed to remove the unwanted catalyst layer regions. It isrecognized that many catalyst layer etch processes may not besignificantly selective in etching the catalyst material over theunderlying metal electrode material. It therefore can be preferred thatthe catalyst etch process be controlled as a timed process or with othercontrols to ensure that the integrity of the metal contact pad materialis maintained.

In an alternate process, the catalyst layer can be patterned and etchedin a sequence of steps separate from that employed for the nanotubeprobe contact pad layer. For example, the contact pad layer can bepatterned by, e.g., a conventional lift-off process as just described,and then the catalyst layer deposited and patterned by a second separatelift-off process. In this scenario, a photoresist layer is formed overthe produced contact pads and patterned to expose regions of the contactpads at which it is desired to provide a region of nanotube catalystlayer. The catalyst layer is then blanket-deposited, preferably by aselected vapor deposition process like that described above. Lift-off ofthe photoresist layer is then carried out to remove portions of thecatalyst layer, resulting in a patterned catalyst region atop thecontact pads.

It is not required that the catalyst layer be patterned by a lift-offprocess; instead, the catalyst layer can be blanket-deposited on thecontact pads and then etched, e.g., by lithographic patterning of aphotoresist layer applied on top of the catalyst layer and patterned todefine distinct catalyst islands. Etching of the catalyst regionsexposed through the photoresist pattern can then be carried out in theconventional manner. This approach, like the catalyst lift-off approach,has the advantage of enabling precise formation of catalyst islands thatdo not necessarily extend across an entire electrode contact pad, andtherefore that more precisely define the location of nanotube synthesis.

Whatever process sequence is employed to produce contact pads andcatalyst regions, it can be preferred in accordance with the inventionto extend the contact pad and catalyst regions across an intendedaperture location, such that production of an aperture through thecontact pad and catalyst layers results in self alignment of the contactpads and catalyst regions with edges of the aperture. This results inproduction of two contact pads that are separated by the aperture. Thecatalyst layer regions can also abut the aperture edge and not extendacross the expanse of the contact pads. Such a condition can be producedby the various catalyst layer etch sequences just described. Whatevercatalyst pattern is desired, it is preferably produced by a lithographicprocess that enables precise definition of the location and extent ofcatalyst regions. This lithographic catalyst definition, in combinationwith vapor deposition of a thin catalyst layer, enables precise nanotubesynthesis.

This lithographic definition of the catalyst regions does not requireetching of the catalyst layer. For example a blanket deposition ofcatalyst layer can be carried out in the manner described above, andthen a capping layer can be deposited and patterned. The capping layerpattern exposes regions of the catalyst layer at which it is desired tosynthesize nanotubes, with the remainder of the catalyst layer beingcovered to inhibit nanotube synthesis. With this configuration, thecatalyst layer is not itself etched, but through lithography the preciselocation of catalyst exposure for nanotube synthesis is accomplished.

Once the catalyst layer regions are formed at selected sites on thecontact pads, an aperture is formed through the membrane or othersupport structure on which nanotubes are to be provided. In one exampleprocess, focused ion beam milling of the catalyst, contact pad, andmembrane materials is carried out directly, in the manner describedpreviously, to enable self alignment of the various layers with theaperture. The resulting structure provides an aperture with contact padsand catalyst regions in alignment. Alternatively, lithographicpatterning of each layer to be etched can be carried out in sequence,with one or more layers etched together as possible by a given etchrecipe. Furthermore, a plurality of apertures can be formed in a givensubstrate, membrane, or other support structure, in arrays or otherconfiguration suitable for a given application.

Once a selected aperture or apertures are produced, nanotube synthesiscan be carried out on the substrate or membrane. The nanotube synthesisis particularly carried out to produce one or more nanotubes bridgingeach aperture to connect to edges of the aperture or to contact pads. Inone example synthesis process, nanotube growth is carried out in asuitable system, e.g., a furnace system. A substrate on which nanotubegrowth is desired is loaded into the furnace system and the temperatureof the system is raised to the desired growth temperature, which can be,e.g., between about 600° C.-1500° C., and preferably is about 900° C.During the temperature ramp, it can be preferred to provide a flow of aninert gas, e.g., argon, to suppress oxidation of the contact padmaterial, catalyst material, membrane and/or substrate material, andother materials included in the configuration.

When the desired synthesis temperature is reached, the gas flow isswitched to a hydrocarbon gas, e.g., a methane gas flow. The methane gasflow is preferably maintained at between about 100 sccm and about 400sccm, with a flow rate of about 200 sccm preferred. With this relativelylow gas flow, it is found in accordance with the invention thatamorphous carbon formation on and around synthesized nanotubes and thesubstrate area is substantially inhibited. As a result, in accordancewith the invention there is no need for inclusion of hydrogen or othergas flow in addition to the methane to inhibit amorphous carbonformation. It is understood in accordance with the invention that theinfluence of gas flow direction on the orientation of nanotubes as theyare synthesized is negligible, and therefore that no particularorientation of substrates with respect to gas flow is required.

The methane gas flow exposure of the catalyst material can be carriedout for any duration required for a given application to producenanotubes of selected diameter and quantity. For many applications, itcan be preferred to carry out the methane gas flow exposure for 10minutes or less to repeatably synthesize single-walled nanotubes. Ifsuch is not a requirement, the gas flow can be continued for anyselected duration corresponding to a desired nanotube wall thickness. Itis found, however, that minimization of nanotube synthesis time can bepreferred in that such reduces the production of amorphous carbon on thenanotubes and surrounding structures.

With this process, nanotubes can be selectively synthesized on thesurface of a support structure at or near to the location of an apertureto be formed into a nanopore in the manner described below. It is to berecognized that electrical characterization of synthesized nanotubes canbe preferable for ensuring that a selected nanotube provides therequisite electrical properties for functioning as a probe. A nanotubewith a desired diameter and electrical properties that has beensynthesized near to a position at a nanopore where the nanotube isdesired can then be manually pushed into position by, e.g., an AFMcantilever tip.

For applications in which a nanotube is to be mounted to the tip of anactuating structure, e.g., the tip of an AFM cantilever beam, fortranslocating a tip-mounted DNA-nanopore complex through a nanopore, theinvention provides a technique for producing a selected nanotube lengthon the mounting tip, as taught in U.S. Ser. No. 11/008,402, Golovchenkoet al., “Patterning by Energetically-Stimulated Local Removal ofSolid-Condensed-Gas Layers and Solid State Chemical Reactions ProducedWith Such Layers,” U.S. patent application publication No. US2007/0262050, the entirety of which is hereby incorporated by reference.This technique can also be applied for producing a desired nanotubelength for operation as a nanotube probe or in a DNA-nanotube hybridcomplex.

In a first step of such a process sequence, a nanotube is provided on ananotube holder that can be provided on a structure holder enablingelectrical and thermal connections for control of the electrical andthermal state of a structure. Preferably the nanotube holder can bemated with the structure holder such that thermal and electricalconnection can be made to the nanotube. The nanotube holder can beprovided as, e.g., an AFM tip, or as another suitable, mechanicallyrigid structure, such as a cantilever structure, on which a nanotube canbe mounted and actuated for controlled translocation through a nanopore,if such is the application to be addressed.

The nanotube can be positioned on the nanotube holder in a number ofways. For many applications, it can be preferred to grow the nanotube insitu on the holder following the technique described above. But anysuitable carbon nanotube synthesis technique can be employed forlocating the carbon nanotube on the nanotube holder as the nanotube isgrown. Alternatively, carbon nanotubes can be synthesized at a locationother than the nanotube holder and then transferred or deposited to theholder. Alternatively, nanotubes can be grown vertically from asubstrate in the conventional manner and then picked up directly onto aholder by bringing a holder into contact with a nanotube at a pointalong the length of the tube. A rigid attachment of the nanotube to theholder can be produced by, e.g., directing an electron beam to theholder to build up a carbon residue that can act as a gluing mechanismbetween the holder and the nanotube. It is recognized that theseattachment techniques can be challenging; therefore, the in situ growthof a nanotube directly on a holder of interest is preferred.

Once a nanotube is located on a nanotube holder, the nanotube and holderare positioned on a structure holder in a processing chamber. A solidice condensate layer is then formed on the nanotube by exposing thenanotube to water vapor at a nanotube temperature of less than about 130K and a local pressure of less than about 10⁻⁴ T. Under theseconditions, a solid ice condensate masking layer having a thickness ofas much as 1 μm can be controllably deposited on the nanotube. It isfound that solid ice condensate masking layer formation can be quitedirectional on a three-dimensional structure such as a nanotube andtherefore that the proximity of the vapor injector at its site on theprocess chamber to the nanotube holder location is preferablyconsidered. The masking layer formation can be monitored in situ by,e.g., SEM imaging of the nanotube as the vapor condensation processprogresses.

Once a masking layer is formed on a nanotube, then an energetic beam isdirected to a location on the masking layer which corresponds to thatpoint along the nanotube that is to be cut for shortening the nanotube.An SEM or other imaging system and technique can be employed for imagingthe nanotube to determine its starting length and to identify the pointat which the tube is to be cut for reducing the tube length. Anenergetic beam, e.g., a 3 KeV, 50 pA electron beam, or an ion beam, isthen directed to that point to locally remove the solid condensatemasking layer just at the location desired for nanotube cutting.

Once local removal of the solid condensate masking layer is complete,thereby exposing a location of the underlying nanotube, then thenanotube itself is cut. Here an energetic beam is directed to thenanotube at the section along the nanotube length that is exposed by thelocal removal of the solid condensate layer, to cut the nanotube to adesired length. The energetic beam employed to cut the nanotube can bethe same as or distinct from the energetic beam employed to locallyremove the solid condensate masking layer. Whatever nanotube cuttingbeam species is employed, the solid condensate masking layer acts toprotect the nanotube and prevent it from bending or moving away from theenergetic beam as the cutting beam species is directed at the exposednanotube section and focused at that exposed section. The solidcondensate masking layer further acts to protect and keep the nanotuberigid as the nanotube is cut at the exposed section. As a result, ahighly focused cutting beam species is not required; the linewidth ofthe locally removed solid condensate masking layer can be employed toset the resolution of the nanotube cutting process.

In an example of such a scenario, an electron beam can be employed tolocally remove a region of a solid ice condensate masking layer on acarbon nanotube, e.g., the 3 KeV, 50 pA beam described above can beemployed to locally remove an ice masking layer formed on a nanotube ata temperature of 128 K and a pressure of 10⁻⁴ T. Then an ion beam can beemployed to cut the nanotube at the location of the nanotube at whichthe ice masking layer was locally removed. For example, a Ga⁺ ion beamof 30 KeV in energy and an amperage of 10 pA can be employed for cuttingthe nanotube. Here the less focused ion beam is employed to cut thenanotube, with the solid ice condensate layer protecting the nanotube.

Once the nanotube is cut, the solid condensate masking layer can beremoved. For many applications, it can be preferred that the maskinglayer be removed by conversion from the solid phase back to the vaporphase. Such vaporization minimizes both the formation of residue on thenanotube and possible damage to the nanotube. In one example process theice condensate masking layer is sublimed by increasing the temperatureof the nanotube to a temperature sufficient for sublimation at processpressures of interest. For example, at a pressure of about 10⁻⁴ T, atemperature of at least about 180 K enables sublimation of an icecondensate layer. Such is found to result in complete removal of the icecondensate layer to the vapor phase, substantially without residue orharm to the easily bent or damaged nanotube. The condensate maskinglayer can be removed by any suitable process as explained previously,including wet chemistries as well as plasma or other vapor processes.With this removal step, a nanotube of a selected length is produced.

The various fabrication processes just described can be employedas-required for producing a selected molecular analysis deviceconfiguration. The following examples demonstrate fabrication techniquesfor producing a range of device configurations.

EXAMPLE I

Referring to FIGS. 8A-8B, in a first example fabrication sequence forproducing an electronic molecular analysis device in accordance with theinvention, a support structure 200 is provided, e.g., a silicon nitridemembrane of about 200 nm in thickness, in the conventional manner, andas taught in U.S. Patent Application Publication US2005/0006224,Golovchenko et al., “Pulsed Ion Beam Control of Solid State Features.”As shown in the top-down planar view of FIG. 8A, the membrane isprovided with a starting aperture 205 by, e.g., electron beam etching,ion beam milling, wet etching, plasma etching, ion beam sculpting, orother suitable process.

In one example, the starting aperture is generally circular, having adiameter of between about, e.g., 50 nm and 100 nm. As depicted in theplanar view of FIG. 8A and the side face view of FIG. 8B, the supportstructure 200 is provided with an upper trench, or groove 208 in the topsurface of the structure and a lower trench 210 in the bottom surface ofthe structure. The upper and lower trenches 208, 210 are orthogonal toeach other, and the aperture 205 is formed at the intersection of thetwo trenches. This orientation enables self-alignment for the nanotubeprobe positioning, as explained below.

The upper and lower trenches can be produced by using a focused ion beamprocess or by standard patterned masking and etching procedures. Formany applications, the trenches can be formed as less than about 100 nmin width and about ½ as deep as the thickness of the support structuremembrane. The exact depth of a trench produced by a given procedure canbe determined by testing a series of stepwise increasingly deeptrenches; e.g., for a 200 nm-thick membrane, trench depths of 50 nm, 70nm, 90 nm, 110 nm, etc., to calibrate the procedure for a given batch ofsilicon nitride membranes. The depth of the trench that produces thedesired diameter starting nanopore, e.g., between about 50 nm and about100 nm, can then be routinely used in an open loop fashion for thesubsequent fabrications.

With this configuration of the support structure complete, a nanotube212 is then provided in the upper trench 208, across the aperture 205.The nanotube 212 can be disposed at the aperture location in the trenchby in situ synthesis of the nanotube at the aperture, or by positioningof a free nanotube at the aperture. In situ synthesis of the nanotubecan be carried out in the manner described above, e.g., with a catalystdeposited and patterned in the upper trench followed by CVD nanotubesynthesis. Alternatively, pre-synthesized nanotubes provided in anon-aqueous solution can be dispensed onto the surface of the supportstructure and a selected nanotube mechanically transported to thetrench.

This mechanical transporting of a selected nanotube has beenexperimentally verified to enable precise movement and positioning to aprespecified location. In one technique for such movement andpositioning, an AFM tip is employed to roll a nanotube across a surfaceand down into a trench to the location of the aperture. Note that forclarity, the features in FIG. 8 are not shown to scale. The nanotubediameter is typically many times smaller than the trench depth andwidth. In addition, surface attraction effects, such as van der Waalsforces, cause a nanotube to tend to remain in place on a surface. As aresult, a nanotube can be well-controlled by an AFM tip and rolledacross a surface and down the wall of a trench into a desired positionat the location of an aperture.

The selected nanotube can be electrically contacted by contact padsformed prior to synthesis, as described above, or subsequentlyelectrically contacted by forming, e.g., palladium contact pads that arein turn connected to larger gold contact pads that connect to off-chipcircuitry by conventional methods, e.g., as in Javey, A., et al. Nature424: 654 (2003) and Javey, A. et al. Nano Letters 4:447 (2004). In thisprocess, palladium is evaporated onto the nanotubes, through a mask, atthe desired location near to the aperture perimeter.

Referring to FIGS. 8C-8D, in a next process step, a selected coating isdeposited on the nanotube-support structure assembly. A particularlywell-suited deposition technique is the atomic layer deposition (ALD)process described above. The ALD process is particularly well-suitedbecause the deposition of material is strictly dependent on the chemicalinteraction between a gas-phase molecule and hydroxyl or otherfunctional groups accessible at the surface of the material on which acoating is to be deposited. Absent such functional groups, no depositionof the gas-phase molecules occur. Carbon nanotubes as-synthesized do notin general possess functionalized surfaces that provide the requisitehydroxyl or other functional groups for deposition of the gas phasematerial. Thus, if an insulating material such as aluminum oxide orhafnium oxide is deposited by ALD on a device configuration thatincludes silicon, silicon nitride and nanotubes, aluminum oxide orhafnium oxide will grow uniformly by chemical reaction at the surfacesof all of these structures, including the newly formed aluminum oxide orhafnium oxide surfaces, but will not grow from the carbon nanotubesurfaces which, as grown, do not have functional groups such as hydroxylgroups at their surface. This condition can be exploited to enablesite-specific deposition of such materials on the support structure butnot directly on, from, or over the unsupported nanotube, e.g., not on orover a region of a nanotube that is suspended over a pore or void. Evenif material deposition does occur on the nanotube, the followingfabrication sequence can be employed.

In one such scenario, a selected number of ALD cycles are carried out,in the manner described above, depositing material on all surfaces ofthe support structure 200 including the walls of the aperture and thetrenches 210. The deposition of the selected material extends over andmay cover regions of a nanotube that are supported by, or proximal toany of the silicon nitride or newly grown ALD material, but does notitself originate from the nanotube surface. As the material depositionis continued, the build up of deposited material at the aperture reducesthe extent of the aperture. Accordingly, the deposition process iscontinued until a selected final nanopore diameter is produced, e.g., adiameter of between about 2 nm and about 10 nm. Due to the very precisenature of the ALD process, the material thickness produced by each ALDcycle can be precisely characterized for a given support structure andnanotube arrangement and dimensions and controlled to achieve a selectedfinal nanopore diameter with the upper side of the nanotube coated. Forexample, with a starting aperture of 50 nm, 220 ALD cycles, each addinga layer 1 Å-thick, would produce a final pore of 6 nm in diameter.

Once a final nanopore diameter is achieved by the ALD process, thenanotube probe configuration is produced at the perimeter of thenanopore. Referring to FIGS. 8E-8F, this configuration is achieved bycutting through the nanotube that lies exposed across the final nanoporeto produce two nanotube ends 216, 218 that abut on the nanoporeperimeter. In one example nanotube cutting technique, an energetic beam220, e.g., a high-energy electron beam or preferably, an ion beam, isdirected through the nanopore, from the bottom or top of the supportstructure. The energetic beam removes the exposed unprotected nanotubematerial from the nanopore, while the aluminum oxide or other ALDcoating protects the ALD covered regions of the nanotube and supportstructure from the beam species. Thus, the nanopore acts as an etchmask, exposing the bare nanotube to be removed from across the nanopore,while the remainder of the structure is protected by the ALD coatingthat is absent within the diameter of the final nanopore. Once the barenanotube is removed from across the final nanopore, a functionalmolecular characterization device like that of FIG. 1A is produced,having the ends of nanotube probes abutting a nanopore perimeter.

EXAMPLE II

Referring now to FIGS. 9A-9C, in a further fabrication sequence forproducing an electronic molecular analysis device, a support structure200 such as a silicon nitride membrane is provided with an aperture 205in the manner of Example I. Orthogonal trenches 208, 210 are provided inthe top and bottom surfaces, respectively, of the support structure,with the aperture located at the intersection of the trenches as inExample I.

With this configuration in place, a nanotube 225 is positioned in thelower trench 225 in the manner of Example I. FIG. 9A depicts the upperplanar surface of the structure, showing the nanotube across theaperture, while FIG. 9C depicts the bottom planar surface of thestructure, showing the nanotube in the bottom trench across theaperture.

As shown in the end face view of FIG. 9B, in a next process step, anenergetic beam 226, e.g., an ion beam, is then directed, from the upperside of the membrane, through the aperture at an angle selected to cutthe nanotube at the position that will leave it abutting the finalnanopore perimeter at the end of the fabrication sequence, e.g., at anangle of about 64 degrees, specifically for the particular geometryshown; for a given geometry, a corresponding angle is to be determined.This angled beam impinges and removes that portion of the nanotube inthe aperture that is in the path of the beam. The resulting structure isshown in FIGS. 9D-F. The portion 228 of the nanotube 225 in the aperturethat was not in the path of the ion beam remains protruding into theaperture.

Referring to FIGS. 9G-I, in a next process step, a second nanotube 230is then provided in the upper trench 208 such that it extends off-centeracross the aperture. This second nanotube 230 can be synthesized in situat the site of the trench or mechanically positioned in the trench andif desired, off-center across the aperture, as in Example I above. Ineither case, if desired, the second nanotube 230 is not positioned inthe center of the aperture but instead, as shown in FIGS. 9G-I, at apoint in the trench such that its side will just abut the perimeter ofthe final nanopore.

With the second nanotube in position, then as shown in FIGS. 9J-L, aselected material is deposited on the support structure, e.g., by ALD inthe manner described above. As in Example I above, the depositedmaterial forms a layer 232 on all of the surfaces except those of theunsupported nanotube, resulting in the structure shown in FIG. 9J.

As shown in FIG. 9K, as the material deposition proceeds, the aperturediameter is reduced by the deposited material. The build up of depositedmaterial also increasingly covers the protruding portion 228 of thefirst nanotube 225 and the upper surface of the second nanotube 230. Thematerial deposition is continued until a selected nanopore diameter isachieved, with the edge of the protruding nanotube portion 228 and thesecond nanotube 230 located at the final nanopore perimeter. To enablethis condition, the selected deposition process, e.g., ALD, ischaracterized for a given support and nanotube arrangement, to ensurethat a desired nanopore diameter is achieved at the point in depositionprocess at which the protruding nanotube portion 228 and the secondnanotube 230 are covered by the material except at a region just at thenanopore perimeter, as shown in FIG. 9L.

With this deposition complete, a fully functional molecularcharacterization device is produced having the arrangement of FIGS.2A-2C. Referring back to FIG. 9I, the nanotube portion 234 of the firstnanotube 225, opposite the protruding portion 228 is not electricallyconnected and does not form part of either circuit 28, 52 shown in FIG.2B. The protruding nanotube portion 228 and the second nanotube 230 formtwo nanotube probes, in an end orientation and a side orientation,respectively, connected in the two circuits for control and analysis ofDNA translocated through the nanopore.

EXAMPLE III

The fabrication sequence of Example II can be extended to produce themolecular analysis device of FIGS. 3A-C and FIG. 4A described above.Referring to FIGS. 10A-C, in this extended process, a nanotube 225 isprovided in the bottom trench 210 of a support membrane 200 having anaperture 205 at the intersection of the bottom trench 210 with an uppertrench 208 as in FIGS. 9A-C. An energetic beam is then employed as inExample II and FIG. 9B to remove a portion of the nanotube 225,resulting in a protrusion 228 of the nanotube into the aperture, as inFIGS. 10A-C. A second nanotube 230 is then positioned, as in FIGS. 9G-Iand 10D-F, at the perimeter of the aperture if desired, in the uppertrench 208 of the support structure.

Referring to FIGS. 10G-I, a third nanotube 234 is then disposed on theupper surface of the support structure 200, orthogonal to the secondnanotube 230 in the upper trench 208 and at a position on the perimeterof the aperture. This third nanotube 234 can be synthesized in situ ormanually positioned at the selected location, both as described above.

In a final process step, referring to FIGS. 10J-L, a selected materialis deposited on the support structure, e.g., by ALD in the mannerdescribed above. The deposited material forms a layer 236 on the uppersurface of the support structure and the upper trench, and does notoriginate at the second nanotube 230, as shown in FIG. 9J. The thirdnanotube 234 remains uncoated in the region of the aperture due to theun-functionalized nanotube surface and the lack of membrane materialaround the third nanotube in the aperture. As a result, the thirdnanotube remains exposed at the perimeter of the aperture.

The deposited material layer 232 also covers the bottom surface of thesupport structure, as shown in FIG. 10L. As shown in FIG. 10K, as thematerial deposition proceeds, the aperture diameter is reduced by thedeposited material. The build up of deposited material also increasinglycovers the protruding portion 228 of the first nanotube 225 and theupper surface of the second nanotube 230. The material deposition iscontinued until a selected nanopore diameter is achieved, with the edgeof the protruding nanotube portion 228 and the second nanotube 230located at the final nanopore perimeter. To enable this condition, theselected deposition process, e.g., ALD, is characterized for a givensupport and nanotube arrangement, to ensure that a desired nanoporediameter is achieved at the point in deposition process at which theprotruding nanotube portion 228 and the second nanotube 230 are coveredby the material except at a region just at the nanopore perimeter, asshown in FIG. 10L.

With the deposition complete, a nanopore having a side-orientedtranslocation control nanotube 234, an end-oriented nanotube probe 228,and a side-oriented nanotube probe 230 are provided. The fabricationsequence of this example results in a translocation control nanotube 234that is orthogonal to the side-oriented nanotube probe 230. In FIG. 4A adevice arrangement is shown in which a translocation control nanotube 65is parallel to a side-oriented nanotube probe 50. Either arrangement canbe employed in accordance with the invention, and this exampledemonstrates that a range of configurations can be obtained withselected fabrication sequences.

If it is desired to employ two end-oriented nanotube probes with atranslocation control nanotube as in the arrangement of FIGS. 3A-3C,then the fabrication sequences of Examples I and III can be combined.Prior to the deposition and nanotube removal steps of FIGS. 8C-8F, thethird nanotube 234 in FIGS. 10G-I can be provided on the top surface ofthe support structure; the deposition and etch steps can then proceed toproduce the structure of FIGS. 3A-3C.

EXAMPLE IV

In a further fabrication sequence for producing a molecular analysisdevice in accordance with the invention, there is provided anend-oriented nanotube probe and a side-oriented nanotube probe disposedalong the length of a nanopore, as in the arrangement of FIG. 4B.Referring to FIGS. 11A-B, in a first step of this fabrication sequence,a relatively thick support structure, e.g., a thick silicon nitridemembrane, is provided. For example, a nitride membrane having athickness of, e.g., about 800 nm can here be employed. A series ofapertures are provided in the membrane in the manner described above.FIG. 11A is a planar top down view of a region of a thick membrane 240in which two apertures 242, 244 are provided. FIG. 11B is a side endface view of the membrane structure of FIG. 11A.

In a next process step, shown in FIGS. 11C-D, an energetic beam 246,e.g., an ion beam, or standard lithographic processing, is used toremove, e.g., about one-third of the membrane material from the top ofthe membrane such that a new membrane surface orthogonal to the originalmembrane surface intersects the diameter of the original membraneapertures. This process is then repeated at the bottom of the membraneto remove, e.g., about one-third of the membrane material from thebottom of the membrane, again creating a new membrane surface orthogonalto the original membrane surface and which intersects the diameter ofthe original membrane apertures. As a result, a portion of the originalmembrane is now only one-third as thick as the original membrane and theother portion of the original membrane remains at its originalthickness. Because the edge of the removed portions of the membrane fromthe top and bottom surface extend through the diameters of the originalapertures, longitudinal grooves 248, 250 are exposed along the length ofthe newly created membrane surfaces that are orthogonal to the originalmembrane surfaces. These groves extend as apertures through thethickness of the residual membrane whose surface is parallel to theoriginal membrane surfaces.

In a next process step, referring to FIGS. 11E-F, nanotubes aresynthesized in situ on the membrane structure, in the manner describedabove. Catalyst material is deposited and patterned adjacent to and inthe groves on one of the newly created orthogonal membrane surfaces andon the residual membrane whose surface is parallel to the originalmembrane surfaces at locations adjacent to the apertures. Nanotubes arethen synthesized on the membrane structure. FIGS. 11E-F depict a numberof synthesized nanotubes, e.g., 252, 254, 256, 258 on the membranesupport structure. Given a number of apertures provided in the membranestructure, it is recognized that at least one synthesized nanotube canbe expected to grow along a grove and through an aperture through thethickness of the residual membrane whose surface is parallel to theoriginal membrane surface, as with the nanotube 256 shown in theaperture 242 of the membrane in FIG. 11E.

With identification of an aperture 242 though which a nanotube 256 hasbeen synthesized, then in a next process step, shown in FIGS. 11G-H, aselected second nanotube 260 is manually positioned relative to theaperture 242 such that an end portion 262 of the second nanotube 260protrudes well into the aperture 242. Alternatively, this secondnanotube could be positioned such that a long side of the nanotube runsoff-center across the aperture, as in Example I above. In this case, thesecond nanotube is not positioned in the center of the aperture butinstead at a position on the membrane surface such that its side willjust abut the perimeter of the final nanopore, as shown for theside-abutting nanotube in Example II. Referring to FIGS. 11I-J, in anext process step, a layer of material 265 is deposited, e.g., by ALD,to coat all of the exposed membrane structures and to reduce theaperture to a final desired nanopore size of, e.g., between about 2 nmand about 10 nm in diameter.

Because it is difficult to manually position the end of the nanotube 260such that its end precisely abuts on the perimeter of the finalnanopore, it may be preferable to initially position the second nanotube260 so that its end protrudes well into and across the diameter ofaperture 242. Because this protruding nanotube end 262 will remainunprotected by the newly deposited ALD material when the final nanoporediameter is produced by ALD, it can be removed with an electron beam,ion beam, or other energetic beam directed through the nanopore, fromthe bottom or top of the support structure. As in example I, theenergetic beam removes the exposed unprotected nanotube material fromthe nanopore, while the aluminum oxide or other ALD coating protects theALD covered regions of the nanotubes and support structure from the beamspecies.

Referring also to FIG. 11M, showing a left-side end face view of themembrane structure, it can be preferred to provide metal contact pads onthe ends of the vertical nanotube 256 in the nanopore 242 prior to thematerial deposition shown in FIGS. 11I-11L. Such metal contact regionsprotect the ends of the vertical nanotube 256 from being covered by thematerial being deposited and put metal connections in place. With thematerial deposition complete, a molecular characterization device likethat of FIG. 4B is produced, having a first nanotube probe verticallyoriented through the length of a nanopore and a second end-orientednanotube probe.

These fabrication examples demonstrate the wide range of processes thatcan be employed to produce many alternative molecular characterizationdevice configurations. The invention does not require a specificfabrication process, however; any suitable manufacturing process forproducing a desired molecular characterization device arrangement can beemployed.

With this discussion, it is shown that the nanopore-based molecularcharacterization instrument of the invention can be applied to a rangeof molecular characterization applications. The molecularcharacterization device can satisfy the requirements for a$1,000/mammalian genome assay because the instrument can directlytransduce a sequence of DNA bases into an electrical signal, on thebasis of their distinct physical and electrical properties, and becausethe instrument enables a single-molecule analysis technique that canachieve about 7.7-fold sequence coverage, or about 6.5-fold coverage inQ20 bases, and over sampling with DNA from <10⁶ target genomes. Thiscorresponds to about 2 nanograms of human genomic material, which can bedirectly obtained without amplification using standard sampling methods.If the identity of each DNA nucleoside base in a sequence of bases isresolved as the bases pass through a nanopore at a rate of about 10⁴bases/second, then an instrument with an array of 100 such nanopores canproduce a high-quality draft sequence of one mammalian genome in about20 hours.

Thus, the high throughput and spatial confinement enabled by thenanopore configuration, in combination with the direct electronicanalysis, molecular orientation control, and molecular translocationspeed control enabled by a nanotube probe configuration, provide for ahigh-bandwidth molecular characterization process that achieves therapid, reliable, and inexpensive molecular analysis and characterizationrequired for a wide range of biological and medical applications. It isrecognized that those skilled in the art may make various modificationsand additions to the embodiments described above without departing fromthe spirit and scope of the present contribution to the art.Accordingly, it is to be understood that the protection sought to beafforded hereby should be deemed to extend to the subject matter claimsand all equivalents thereof fairly within the scope of the invention.

1. A molecular characterization device comprising: a first reservoircontaining a liquid solution including a molecule to be characterized; asecond reservoir for containing a liquid solution including a moleculethat has been characterized; a solid state support structure includingan aperture having a molecular entrance providing a fluidic connectionto the first reservoir and a molecular exit providing a fluidicconnection to the second reservoir; one carbon nanotube having alongitudinal sidewall disposed as a molecular contacting surface at theaperture; a voltage source connected in series with the carbon nanotubefor electrically biasing the carbon nanotube; and an electrical currentmonitor connected in series with the carbon nanotube for monitoringchanges in electrical current through the nanotube corresponding totranslocation of a molecule through the aperture.
 2. The molecularcharacterization device of claim 1 wherein the carbon nanotube isdisposed under an electrically insulating material layer betweenlongitudinal ends of the nanotube except along the carbon nanotubemolecular contacting surface at the aperture.
 3. The molecularcharacterization device of claim 1 wherein the aperture is coated withan electrically insulating material layer.
 4. The molecularcharacterization device of claim 1 wherein the electrical currentmonitor comprises an ammeter.
 5. The molecular characterization deviceof claim 1 wherein the voltage source is controllably connected to applyto the carbon nanotube a sequence of voltage biases, having alternatingpolarity, for sequentially attracting and repelling molecules to themolecular contacting surface at the aperture.
 6. The molecularcharacterization device of claim 5 wherein the sequence of carbonnanotube voltage biases that the voltage source is connected to applyincludes a positive bias with respect to the liquid solution forattracting a molecule to the molecular contacting surface at theaperture.
 7. The molecular characterization device of claim 5 whereinthe sequence of carbon nanotube voltage biases that the voltage sourceis connected to apply includes a negative bias with respect to theliquid solution for repelling a molecule from the molecular contactingsurface at the aperture.
 8. The molecular characterization device ofclaim 1 wherein the electrical current monitor is connected formonitoring changes in electrical conductance of the carbon nanotube. 9.The molecular characterization device of claim 1 wherein the electricalcurrent monitor is connected for monitoring changes in electricalmobility of the carbon nanotube.
 10. The molecular characterizationdevice of claim 1 wherein the molecule comprises a nucleoside base thatcan couple to the molecular contacting surface at the aperture whilebeing translocated through the aperture.
 11. The molecularcharacterization device of claim 1 wherein the molecule to becharacterized comprises a biomolecule.
 12. The molecularcharacterization device of claim 1 wherein the molecule comprises apolymer molecule.
 13. The molecular characterization device of claim 1wherein the molecule comprises a biopolymer molecule.
 14. The molecularcharacterization device of claim 13 wherein the molecule is selectedfrom the group consisting of proteins, polynucleic acids, DNA, and RNA.15. The molecular characterization device of claim 1 wherein themolecule to be characterized comprises a molecule coupled to a carbonnanotube.
 16. The molecular characterization device of claim 15 whereinthe molecule coupled to a carbon nanotube comprises at least one strandof DNA coupled to a carbon nanotube.
 17. The molecular characterizationdevice of claim 15 wherein the molecule coupled to a carbon nanotubecomprises at least one strand of RNA coupled to a carbon nanotube. 18.The molecular characterization device of claim 15 wherein the carbonnanotube to which the molecule is coupled is connected to a cantileveredactuation tip for translocation of the nanotube through the aperture.19. The molecular characterization device of claim 1 wherein the carbonnanotube comprises a semiconducting nanotube.
 20. The molecularcharacterization device of claim 1 wherein the carbon nanotube comprisesa metallic nanotube.
 21. The molecular characterization device of claim1 wherein the carbon nanotube sidewall abuts a perimeter of theaperture.
 22. The molecular characterization device of claim 1 whereinthe aperture comprises a pore, extending through the thickness of thesupport structure, and having a diameter sufficiently small to permitonly a single molecule at a time to be translocated through the pore.23. The molecular characterization device of claim 22 wherein the porecomprises a nanopore having a diameter less than about 100 nm.
 24. Themolecular characterization device of claim 22 wherein the nanopore has adiameter less than about 50 nm.
 25. The molecular characterizationdevice of claim 22 wherein the nanopore has a diameter less than about20 nm.
 26. The molecular characterization device of claim 22 wherein thenanopore has a diameter less than about 10 nm.
 27. The molecularcharacterization device of claim 1 wherein the support structurecomprises an electrically insulating membrane in which the aperture isprovided.
 28. The molecular characterization device of claim 27 whereinthe membrane comprises a silicon nitride membrane.